Polynucleotide compositions encoding broad spectrum delta-endotoxins

ABSTRACT

Disclosed are novel synthetically-modified  B. thuringiensis  chimeric crystal proteins having improved insecticidal activity and broader insect host range against coleopteran, dipteran and lepidopteran insects. Also disclosed are the nucleic acid segments encoding these novel peptides. Methods of making and using these genes and proteins are disclosed as well as methods for the recombinant expression, and transformation of suitable host cells. Transformed host cells and transgenic plants expressing the modified endotoxin are also aspects of the invention.

1. BACKGROUND OF THE INVENTION

[0001] The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/253,341, filed Feb. 19, 1999, which is acontinuation of U.S. patent application Ser. No. 08/922,505, filed Sep.3, 1997, which is a continuation-in-part of U.S. patent application Ser.No. 08/754,490, filed Nov. 20, 1996, now U.S. Pat. No. 6,017,534; theentire contents of each is herein incorporated by reference.

1.1 FIELD OF THE INVENTION

[0002] The present invention provides new proteins for combatinginsects, and particularly, coleopteran, dipteran, and lepidopteraninsects sensitive to the disclosed δ-endotoxins derived from Bacillusthuringiensis. The invention provides novel chimeric crystal proteinsand the chimeric cry gene segments which encode them, as well as methodsfor making and using these DNA segments, methods of producing theencoded proteins, methods for making synthetically-modified chimericcrystal proteins, and methods of making and using the synthetic crystalproteins.

1.2 DESCRIPTION OF RELATED ART 1.2.1 B. Thuringiensis Crystal Proteins

[0003] The Gram-positive soil bacterium B. thuringiensis is well knownfor its production of proteinaceous parasporal crystals, orδ-endotoxins, that are toxic to a variety of lepidopteran, coleopteran,and dipteran larvae. B. thuringiensis produces crystal proteins duringsporulation which are specifically toxic to certain species of insects.Many different strains of B. thuringiensis have been shown to produceinsecticidal crystal proteins, and compositions comprising B.thuringiensis strains which produce proteins having insecticidalactivity have been used commercially as environmentally-acceptableinsecticides because of their toxicity to the specific target insect,and non-toxicity to plants and other non-targeted organisms.

[0004] Commercial formulations of naturally occurring B. thuringiensisisolates have long been used for the biological control of agriculturalinsect pests. In commercial production, the spores and crystals obtainedfrom the fermentation process are concentrated and formulated for foliarapplication according to conventional agricultural practices.

1.2.2 Nomenclature of Crystal Proteins

[0005] A review by Höfte et al., (1989) describes the general state ofthe art with respect to the majority of insecticidal B. thuringiensisstrains that have been identified which are active against insects ofthe Order Lepidoptera, i.e., caterpillar insects. This treatise alsodescribes B. thuringiensis strains having insecticidal activity againstinsects of the Orders Diptera (i.e. flies and mosquitoes) and Coleoptera(i.e. beetles). A number of genes encoding crystal proteins have beencloned from several strains of B. thuringiensis. Höfte et al. (1989)discusses the genes and proteins that were identified in B.thuringiensis prior to 1990, and sets forth the nomenclature andclassification scheme which has traditionally been applied to B.thuringiensis genes and proteins. cry1 genes encode lepidopteran-toxicCry1 proteins. cry2 genes encode Cry2 proteins that are toxic to bothlepidopterans and dipterans. cry3 genes encode coleopteran-toxic Cry3proteins, while cry4 genes encode dipteran-toxic Cry4 proteins, etc.

[0006] Recently a new nomenclature has been proposed whichsystematically classifies the Cry proteins based upon amino acidsequence homology rather than upon insect target specificity. Thisclassification scheme is summarized and regularly updated in a databasemaintained by the Bacillus thuringiensis Delta-Endotoxin NomenclatureCommittee at the following web site address:

[0007] http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html.

1.2.3 Mode of Crystal Protein Toxicity

[0008] All δ-endotoxin crystals are toxic to insect larvae by ingestion.Solubilization of the crystal in the midgut of the insect releases theprotoxin form of the δ-endotoxin which, in most instances, issubsequently processed to an active toxin by midgut protease. Theactivated toxins recognize and bind to the brush-border of the insectmidgut epithelium through receptor proteins. Several putative crystalprotein receptors have been isolated from certain insect larvae (Knightet al., 1995; Gill et al., 1995; Masson et al., 1995). The binding ofactive toxins is followed by intercalation and aggregation of toxinmolecules to form pores within the midgut epithelium. This process leadsto osmotic imbalance, swelling, lysis of the cells lining the midgutepithelium, and eventual larvae mortality.

1.2.4 Molecular Biology of δ-Endotoxins

[0009] With the advent of molecular genetic techniques, variousδ-endotoxin genes have been isolated and their DNA sequences determined.These genes have been used to construct certain genetically engineeredB. thuringiensis products that have been approved for commercial use.Recent developments have seen new δ-endotoxin delivery systemsdeveloped, including plants that contain and express geneticallyengineered δ-endotoxin genes.

[0010] The cloning and sequencing of a number of δ-endotoxin genes froma variety of Bacillus thuringiensis strains have been described and aresummarized by Höfte and Whiteley, 1989. Plasmid shuttle vectors designedfor the cloning and expression of δ-endotoxin genes in E. coli or B.thuringiensis are described by Gawron-Burke and Baum (1991). U.S. Pat.No. 5,441,884 discloses a site-specific recombination system forconstructing recombinant B. thuringiensis strains containing δ-endotoxingenes that are free of DNA not native to B. thuringiensis.

[0011] The Cry1 family of crystal proteins, which are primarily activeagainst lepidopteran pests, are the best studied class of δ-endotoxins.The pro-toxin form of Cry1 δ-endotoxins consist of two approximatelyequal sized segments. The carboxyl-half, or pro-toxin segment, is nottoxic and is thought to be important for crystal formation (Arvidson etal., 1989). The amino-half of the protoxin comprises the active-toxinsegment of the Cry1 molecule and may be further divided into threestructural domains as determined by the recently describedcrystallographic structure for the active toxin segment of the Cry1Aaδ-endotoxin (Grochulski et al., 1995). Domain 1 occupies the first thirdof the active toxin and is essential for channel formation (Thompson etal., 1995). Domain 2 and domain 3 occupy the middle and last third ofthe active toxin, respectively. Both domains 2 and 3 have beenimplicated in receptor binding and insecticidal host range activity,depending on the insect and 6-endotoxin being examined (Thompson et al.,1995).

1.2.5 Chimeric Crystal Proteins

[0012] In recent years, researchers have focused effort on theconstruction of hybrid δ-endotoxins with the hope of producing proteinswith enhanced activity or improved properties. Advances in the art ofmolecular genetics over the past decade have facilitated a logical andorderly approach to engineering proteins with improved properties.Site-specific and random mutagenesis methods, the advent of polymerasechain reaction methodologies, and the development of recombinant methodsfor generating gene fusions and constructing chimeric proteins havefacilitated an assortment of methods for changing amino acid sequencesof proteins, fusing portions of two or more proteins together in asingle recombinant protein, and altering genetic sequences that encodeproteins of commercial interest.

[0013] Unfortunately, for crystal proteins, these techniques have onlybeen exploited in limited fashion. The likelihood of arbitrarilycreating a chimeric protein with enhanced properties from portions ofthe numerous native proteins which have been identified is remote giventhe complex nature of protein structure, folding, oligomerization,activation, and correct processing of the chimeric protoxin to an activemoiety. Only by careful selection of specific target regions within eachprotein, and subsequent protein engineering can toxins be synthesizedwhich have improved insecticidal activity.

[0014] Some success in the area, however, has been reported in theliterature. For example, the construction of a few hybrid δ-endotoxinsis reported in the following related art:

[0015] Intl. Pat. Appl. Publ. No. WO 95/30753 discloses the constructionof hybrid B. thuringiensis δ-endotoxins for production in Pseudomonasfluorescens in which the non-toxic protoxin fragment of Cry1F has beenreplaced by the non-toxic protoxin fragment from the Cry1Ac/Cry1Ab thatis disclosed in U.S. Pat. No. 5,128,130.

[0016] U.S. Pat. No. 5,128,130 discloses the construction of hybrid B.thuringiensis δ-endotoxins for production in P. fluorescens in which aportion of the non-toxic protoxin segment of Cry1Ac is replaced with thecorresponding non-toxic protoxin fragment of Cry1Ab.

[0017] U.S. Pat. No. 5,055,294 discloses the construction of a specifichybrid δ-endotoxin between Cry1Ac (amino acid residues 1-466) and Cry1Ab(amino acid residues 466-1155) for production in P. fluorescens.Although the aforementioned patent discloses the construction of ahybrid toxin within the active toxin segment, no specifics are presentedin regard to the hybrid toxin's insecticidal activity.

[0018] Intl. Pat. Appl. Publ. No. WO 95/30752 discloses the constructionof hybrid B. thuringiensis δ-endotoxins for production in P. fluorescensin which the non-toxic protoxin segment of Cry1C is replaced by thenon-toxic protoxin segment from Cry1Ab. The aforementioned applicationfurther discloses that the activity against Spodoptera exigua for thehybrid δ-endotoxin is improved over that of the parent active toxin,Cry1C.

[0019] Intl. Pat. Appl. Publ. No. WO 95/06730 discloses the constructionof a hybrid B. thuringiensis δ-endotoxin consisting of domains 1 and 2of Cry1E coupled to domain 3 and the non-toxic protoxin segment ofCry1C. Insect bioassays performed against Manduca sexta (sensitive toCry1C and Cry1E), Spodoptera exigua (sensitive to Cry1C), and Mamestrabrassicae (sensitive to Cry1C) show that the hybrid Cry1E/Cry1C hybridtoxin is active against M. sexta, S. exigua, and M. brassicae. Thebioassay results were expressed as EC₅₀ values (toxin concentrationgiving a 50% growth reduction) rather than LC₅₀ values (toxinconcentration giving 50% mortality). Although the δ-endotoxins used forbioassay were produced in B. thuringiensis, only artificially-generatedactive segments of the δ-endotoxins were used, not thenaturally-produced crystals typically produced by B. thuringiensis thatare present in commercial B. thuringiensis formulations. Bioassayresults indicated that the LC₅₀ values for the hybrid Cry1E/Cry1Ccrystal against S. frugiperda were 1.5 to 1.7 fold lower (more active)than for native Cry1C. This art also discloses the construction of ahybrid B. thuringiensis δ-endotoxin between Cry1Ab (domains 1 and 2) andCry1C (domain 3 and the non-toxic protoxin segment), although no dataare given regarding the hybrid toxin's activity or usefulness.

[0020] Lee et al. (1995) report the construction of hybrid B.thuringiensis δ-endotoxins between Cry1Ac and Cry1Aa within the activetoxin segment. Artificially generated active segments of the hybridtoxins were used to examine protein interactions in susceptible insectbrush border membranes vesicles (BBMV). The bioactivity of the hybridtoxins was not reported.

[0021] Honee et al. (1991) report the construction of hybridδ-endotoxins between Cry1C (domain 1) and Cry1Ab (domains 2 and 3) andthe reciprocal hybrid between Cry1Ab (domain 1) and Cry1C (domains 2 and3). These hybrids failed to show any significant increase in activityagainst susceptible insects. Furthermore, the Cry1C (domain 1)/Cry1Ab(domains 2 and 3) hybrid toxin was found to be hypersensitive toprotease degradation. A report by Schnepf et al. (1990) discloses theconstruction of Cry1Ac hybrid toxin in which a small portion of domain 2was replaced by the corresponding region of Cry1Aa, although nosignificant increase in activity against susceptible insect larvae wasobserved.

1.3 Deficiencies in the Prior Art

[0022] There exists a need in the art for new methods and compositionscomprising recombinant crystal proteins that exhibit increasedinsecticidal activity and broader-host-range activity.

2. SUMMARY OF THE INVENTION

[0023] The present invention provides novel chimeric δ-endotoxins havingimproved insecticidal activity and broader host-range activity.

[0024] Disclosed are methods for the construction of B. thuringiensishybrid δ-endotoxins comprising amino acid sequences from native Cry1Acand Cry1F crystal proteins. These hybrid proteins, in which all or aportion of Cry1Ac domain 2, all or a portion of Cry1Ac domain 3, and allor a portion of the Cry1Ac protoxin segment is replaced by thecorresponding portions of Cry1F, possess not only the insecticidalcharacteristics of the parent δ-endotoxins, but also have the unexpectedproperties of broader insect host-range and increased insecticidalactivity, relative to the native δ-endotoxins from which the chimericproteins were engineered.

[0025] Specifically, the present invention discloses and claimsgenetically-engineered hybrid δ-endotoxins which comprise a portion of aCry1Ac crystal protein fused to a portion of a Cry1F crystal protein.These chimeric endotoxins have activity against a broader range ofinsects pests described herein.

[0026] In a further embodiment, the present invention also discloses andclaims recombinant B. thuringiensis hybrid δ-endotoxins which comprise aportion of Cry1Ab, Cry1F, and Cry1Ac in which all or a portion of Cry1Abdomain 2 or all or a portion of Cry1Ab domain 3 is replaced by thecorresponding portions of Cry1F and all or a portion of the Cry1Abprotoxin segment is replaced by the corresponding portions of Cry1Ac.Exemplary hybrid δ-endotoxins between Cry1Ab and Cry1F are identified inSEQ ID NO:13 and SEQ ID NO:14.

[0027] One aspect of the present invention demonstrates the unexpectedresult that certain hybrid δ-endotoxins derived from Cry1Ac and Cry1Fproteins exhibit not only the insecticidal characteristics of the parentδ-endotoxins, but also possess insecticidal activity which is notproficiently displayed by either of the parent δ-endotoxins.

[0028] Another aspect of the invention further demonstrates theunexpected result that certain chimeric Cry1Ab/Cry1F proteins maintainnot only the insecticidal characteristics of the parent δ-endotoxins,but also exhibit insecticidal activity which is not displayed by eitherthe native Cry1Ab or Cry1F endotoxins.

[0029] The present invention also encompasses Cry1Ac/Cry1F andCry1Ab/Cry1F hybrid δ-endotoxins that maintain the desirablecharacteristics needed for commercial production in B. thuringiensis.Specifically, the hybrid δ-endotoxins identified in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQID NO:34 can efficiently form proteinaceous parasporal inclusions in B.thuringiensis and have the favorable characteristics of solubility,protease susceptibility, and insecticidal activity of the parentδ-endotoxins.

[0030] In a further embodiment, the present invention also discloses andclaims recombinant B. thuringiensis hybrid δ-endotoxins which comprise aportion of Cry1Ac and Cry1C in which all or a portion of Cry1Ac domain 3is replaced by the corresponding portions of Cry1C and all or a portionof the Cry1Ac protoxin segment is replaced by the corresponding portionof Cry1C. Exemplary hybrid δ-endotoxins between Cry1Ac and Cry1C areidentified in SEQ ID NO:29 and SEQ ID NO:30.

[0031] One aspect of the present invention demonstrates the unexpectedresult that, although neither Cry1Ac nor Cry1C possess S. frugiperdaactivity, the Cry1Ac/Cry1C hybrid δ-endotoxin identified by SEQ ID NO:29and SEQ ID NO:30 has significant activity against S. frugiperda.Furthermore, the Cry1Ac/Cry1C hybrid δ-endotoxin identified by SEQ IDNO:29 and SEQ ID NO:30 has significantly better activity against S.exigua than the Cry1C parental δ-endotoxin.

[0032] The present invention further pertains to the recombinant nucleicacid sequences which encode the novel crystal proteins disclosed herein.Specifically, the invention discloses and claims the nucleic acidsequences of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, and SEQ ID NO:33; nucleic acid sequences whichare complementary to the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29; and SEQID NO:33, and nucleic acid sequences which hybridize to the sequences ofSEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQID NO:29, and SEQ ID NO:33.

[0033] The novel hybrid δ-endotoxins disclosed herein are useful in thecontrol of a broad range of insect pests. The host range of the novelhybrid δ-endotoxins preferably encompasses Coleopteran, Dipteran and/orLepidopteran insects. Of particular interest are boll weevil and wormspecies of Heliothis, Helicoverpa, Pectinophora, Spodotera, and Earias.Such species include, but are not limited to, Heliothis virescens,Helicoverpa zea, Helicoverpa armigera, Pectinophora gossypiella,Spodoptera exigua, Spodoptera frugiperda, Earias vitella, and Spodopteralitura.

[0034] The hybrid δ-endotoxins are described in FIG. 1 and FIG. 4 andare disclosed in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:34. The nucleic acid segmentsencoding these proteins are disclosed in SEQ ID NO:9, SEQ ID NO:11, SEQID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:33.

[0035] The broad host range of the improved δ-endotoxins specified inthe present invention is useful in circumventing dilution effects causedby expressing multiple δ-endotoxin genes within a single B.thuringiensis strain. Expression of such a broad host range δ-endotoxinin plants is expected to impart protection against a wider variety ofinsect pests.

[0036] The impetus for constructing these and other hybrid δ-endotoxinsis to create novel toxins with increased insecticidal activity, broaderinsect host-range, and improved production characteristics. The DNAsequences listed in Table 6 define the exchange points for the hybridδ-endotoxins pertinent to the present invention and as oligonucleotideprimers, may be used to identify like or similar hybrid δ-endotoxins bySouthern or colony hybridization under conditions of moderate to highstringency. Researchers skilled in the art will recognize the importanceof the exchange site chosen between two or more δ-endotoxins can beachieved using a number of in vivo or in vitro molecular genetictechniques. Small variations in the exchange region between two or moreδ-endotoxins may yield similar results or, as demonstrated for EG11062and EG11063, adversely affect desirable traits. Similarly, largevariations in the exchange region between two or more δ-endotoxins mayhave no effect on desired traits, as demonstrated by EG11063 andEG11074, or may adversely affect desirable traits, as demonstrated byEG11060 and EG11063.

[0037] Favorable traits with regard to improved insecticidal activity,increased host range, and improved production characteristics may beachieved by other such hybrid δ-endotoxins including, but not limitedto, the cry1, cry2, cry3, cry4, cry5, cry6, cry7, cry8, cry9, cry10,cry11, cry12, cry13, cry14, cry15 class of δ-endotoxin genes and the B.thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes ofB. thuringiensis insecticidal proteins include, but are not limited toCry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba, Cry1Bb, Cry1Ca, Cry1Cb,Cry1Da, Cry1Db, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Ha, Cry2a,Cry2b, Cry1Ja, Cry1Ka, Cry11Aa, Cry11Ab, Cry12Aa, Cry3Ba, Cry3Bb, Cry3C,Cry4a, Cry4Ba, Cry5a, Cry5Ab, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry8Aa,Cry8Ba, Cry8Ca, Cry9Aa, Cry9Ba, Cry9Ca, Cry10Aa, Cry11Aa, Cry12Aa,Cry13Aa, Cry14Aa, Cry15Aa, Cyt1Aa, and Cyt2Aa. Related hybridδ-endotoxins would consist of the amino portion of one of theaforementioned δ-endotoxins, including all or part of domain 1 or domain2, fused to all or part of domain 3 from another of the aforementionedδ-endotoxins. The non-active protoxin fragment of such hybridδ-endotoxins may consist of the protoxin fragment from any of theaforementioned δ-endotoxins which may act to stabilize the hybridδ-endotoxin as demonstrated by EG11087 and EG11091 (see e.g., Table 3).Hybrid δ-endotoxins possessing similar traits as those described in thepresent invention could be constructed by conservative, or “similar”replacements of amino acids within hybrid δ-endotoxins. Suchsubstitutions would mimic the biochemical and biophysical properties ofthe native amino acid at any position in the protein. Amino acidsconsidered similar include for example, but are not limited to:

[0038] Ala, Ser, and Thr;

[0039] Asp and Glu;

[0040] Asn and Gln;

[0041] Lys and Arg;

[0042] Ile, Leu, Met, and Val; and

[0043] Phe, Tyr, and Trp.

[0044] Researchers skilled in the art will recognize that improvedinsecticidal activity, increased host range, and improved productioncharacteristics imparted upon hybrid δ-endotoxins may be furtherimproved by altering the genetic code for one or more amino acidpositions in the hybrid δ-endotoxin such that the position, orpositions, is replaced by any other amino acid. This may be accomplishedby targeting a region or regions of the protein for mutagenesis by anynumber of established mutagenic techniques, including those proceduresrelevant to the present invention. Such techniques include site-specificmutagenesis (Kunkle, 1985; Kunkle et al., 1987), DNA shuffling (Stemmer,1994), and PCR™ overlap extension (Horton et al., 1989). Since aminoacids situated at or near the surface of a protein are likelyresponsible for its interaction with other proteinaceous ornon-proteinaceous moieties, they may serve as “target” regions formutagenesis. Such surface exposed regions may consist of, but not belimited to, surface exposed amino acid residues within the active toxinfragment of the protein and include the inter-α-helical orinter-β-strand “loop”-regions of δ-endotoxins that separate α-heliceswithin domain 1 and β-strands within domain 2 and domain 3. Suchprocedures may favorably change the protein's biochemical andbiophysical characteristics or its mode of action as outlined in theSection 1. These include, but are not limited to: 1) improved crystalformation, 2) improved protein stability or reduced proteasedegradation, 3) improved insect membrane receptor recognition andbinding, 4) improved oligomerization or channel formation in the insectmidgut endothelium, and 5) improved insecticidal activity orinsecticidal specificity and/or 6) broader insect host-range, due to anyor all of the reasons stated above.

2.1 Crystal Protein Transgenes and Transgenic Plants

[0045] In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel chimeric crystal proteins of the present invention.The process of producing transgenic plants is well-known in the art. Ingeneral, the method comprises transforming a suitable host cell with aDNA segment which contains a promoter operatively linked to a codingregion that encodes a B. thuringiensis Cry1Ac-1F or Cry1Ab-1F,Cry1Ac-1C, or a Cry1Ab-1Ac-1F chimeric crystal protein. Such a codingregion is generally operatively linked to a transcription-terminatingregion, whereby the promoter is capable of driving the transcription ofthe coding region in the cell, and hence providing the cell the abilityto produce the recombinant protein in vivo. Alternatively, in instanceswhere it is desirable to control, regulate, or decrease the amount of aparticular recombinant crystal protein expressed in a particulartransgenic cell, the invention also provides for the expression ofcrystal protein antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

[0046] Another aspect of the invention comprises a transgenic plantwhich express a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression. The construction and expression of synthetic B.thuringiensis genes in plants has been described in detail in U.S. Pat.Nos. 5,500,365 and 5,380,831 (each specifically incorporated herein byreference).

[0047] It is contemplated that in some instances the genome of atransgenic plant of the present invention will have been augmentedthrough the stable introduction of one or more cry1Ac-1F, cry1Ab-1F,cry1Ac-1C, or cry1Ab-1Ac-1F transgenes, either native,synthetically-modified, or further mutated. In some instances, more thanone transgene will be incorporated into the genome of the transformedhost plant cell. Such is the case when more than one crystalprotein-encoding DNA segment is incorporated into the genome of such aplant. In certain situations, it may be desirable to have one, two,three, four, or even more B. thuringiensis crystal proteins (eithernative or recombinantly-engineered) incorporated and stably expressed inthe transformed transgenic plant.

[0048] A preferred gene, such as those disclosed in SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQID NO:33 which may be introduced includes, for example, a crystalprotein-encoding a DNA sequence from bacterial origin, and particularlyone or more of those described herein which are obtained from Bacillusspp. Highly preferred nucleic acid sequences are those obtained from B.thuringiensis, or any of those sequences which have been geneticallyengineered to decrease or increase the insecticidal activity of thecrystal protein in such a transformed host cell.

[0049] Means for transforming a plant cell and the preparation of atransgenic cell line are well-known in the art, and are discussedherein. Vectors, plasmids, cosmids, yeast artificial chromosomes (YACs)and nucleic acid segments for use in transforming such cells will, ofcourse, generally comprise either the operons, genes, or gene-derivedsequences of the present invention, either native, orsynthetically-derived, and particularly those encoding the disclosedcrystal proteins. These DNA constructs can further include structuressuch as promoters, enhancers, polylinkers, or even gene sequences whichhave positively- or negatively-regulating activity upon the particulargenes of interest as desired. The DNA segment or gene may encode eithera native or modified crystal protein, which will be expressed in theresultant recombinant cells, and/or which will impart an improvedphenotype to the regenerated plant. Nucleic acid sequences optimized forexpression in plants have been disclosed in Intl. Pat. Appl. Publ. No.WO 93/07278 (specifically incorporated herein by reference).

[0050] Such transgenic plants may be desirable for increasing theinsecticidal resistance of a monocotyledonous or dicotyledonous plant,by incorporating into such a plant, a transgenic DNA segment encodingCry1Ac-1F and/or Cry1Ac-1C, and/or Cry1Ab-1F and/or Cry1Ab-1Ac-1Fcrystal protein(s) which possess increased insecticidal activity and/orinsecticidal activity over a broader insect host-range. Particularlypreferred plants such as grains, including but not limited to corn,wheat, oats, rice, maize, and barley; cotton; soybeans and otherlegumes; trees, including but not limited to ornamentals, shrubs,fruits, nuts; vegetables, turf and pasture grasses, berries, citrus, andother crops of commercial interest; such as garden crops and/orhouseplants, succulents, cacti, and flowering species.

[0051] In a related aspect, the present invention also encompasses aseed produced by the transformed plant, a progeny from such seed, and aseed produced by the progeny of the original transgenic plant, producedin accordance with the above process. Such progeny and seeds will have astably crystal protein transgene stably incorporated into its genome,and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more chimeric crystal proteins or polypeptidesare aspects of this invention.

2.2 Crystal Protein Screening and Immunodetection Kits

[0052] The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides.Exemplary proteins include those disclosed in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQID NO:34. Said kit can contain a nucleic acid segment or an antibody ofthe present invention. The kit can contain reagents for detecting aninteraction between a sample and a nucleic acid or antibody of thepresent invention. The provided reagent can be radio-, fluorescently- orenzymatically-labeled. The kit can contain a known radiolabeled agentcapable of binding or interacting with a nucleic acid or antibody of thepresent invention.

[0053] The reagent of the kit can be provided as a liquid solution,attached to a solid support or as a dried powder. Preferably, when thereagent is provided in a liquid solution, the liquid solution is anaqueous solution. Preferably, when the reagent provided is attached to asolid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent provided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

[0054] In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

[0055] In general, the detection of immunocomplex formation is quitewell known in the art and may be achieved through the application ofnumerous approaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

[0056] For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

[0057] The container will generally include a vial into which theantibody, antigen or detection reagent may be placed, and preferablysuitably aliquotted. The kits of the present invention will alsotypically include a means for containing the antibody, antigen, andreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

2.3 ELISAs and Immunoprecipitation

[0058] ELISAs may be used in conjunction with the invention. In an ELISAassay, proteins or peptides incorporating crystal protein antigensequences are immobilized onto a selected surface, preferably a surfaceexhibiting a protein affinity such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbedmaterial, it is desirable to bind or coat the assay plate wells with anonspecific protein that is known to be antigenically neutral withregard to the test antisera such as bovine serum albumin (BSA), caseinor solutions of milk powder. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

[0059] After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hours, at temperatures preferably on theorder of about 25° to about 27° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

[0060] Following formation of specific immunocomplexes between the testsample and the bound antigen, and subsequent washing, the occurrence andeven amount of immunocomplex formation may be determined by subjectingsame to a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween®).

[0061] After incubation with the second enzyme-tagged antibody, andsubsequent to washing to remove unbound material, the amount of label isquantified by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonicacid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label.Quantitation is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

[0062] The anti-crystal protein antibodies of the present invention areparticularly useful for the isolation of other crystal protein antigensby immunoprecipitation. Immunoprecipitation involves the separation ofthe target antigen component from a complex mixture, and is used todiscriminate or isolate minute amounts of protein. For the isolation ofmembrane proteins cells must be solubilized into detergent micelles.Nonionic salts are preferred, since other agents such as bile salts,precipitate at acid pH or in the presence of bivalent cations.

[0063] In an alternative embodiment the antibodies of the presentinvention are useful for the close juxtaposition of two antigens. Thisis particularly useful for increasing the localized concentration ofantigens, e.g. enzyme-substrate pairs.

2.4 Western Blots

[0064] The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

[0065] Immunologically-based detection methods for use in conjunctionwith Western blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

2.5 Epitopic Core Sequences

[0066] The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies. In particular, the invention concerns epitopic coresequences derived from Cry proteins or peptides.

[0067] As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a crystal protein or polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the crystal protein orpolypeptide will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

[0068] The identification of Cry immunodominant epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

[0069] Preferred peptides for use in accordance with the presentinvention will generally be on the order of about 8 to about 20 aminoacids in length, and more preferably about 8 to about 15 amino acids inlength. It is proposed that shorter antigenic crystal protein-derivedpeptides will provide advantages in certain circumstances, for example,in the preparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

[0070] It is proposed that particular advantages of the presentinvention may be realized through the preparation of synthetic peptideswhich include modified and/or extended epitopic/immunogenic coresequences which result in a “universal” epitopic peptide directed tocrystal proteins, and in particular Cry and Cry-related sequences. Theseepitopic core sequences are identified herein in particular aspects ashydrophilic regions of the particular polypeptide antigen. It isproposed that these regions represent those which are most likely topromote T-cell or B-cell stimulation, and, hence, elicit specificantibody production.

[0071] An epitopic core sequence, as used herein, is a relatively shortstretch of amino acids that is “complementary” to, and therefore willbind, antigen binding sites on the crystal protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

[0072] In general, the size of the polypeptide antigen is not believedto be particularly crucial, so long as it is at least large enough tocarry the identified core sequence or sequences. The smallest usefulcore sequence anticipated by the present disclosure would generally beon the order of about 8 amino acids in length, with sequences on theorder of 10 to 20 being more preferred. Thus, this size will generallycorrespond to the smallest peptide antigens prepared in accordance withthe invention. However, the size of the antigen may be larger wheredesired, so long as it contains a basic epitopic core sequence.

[0073] The identification of epitopic core sequences is known to thoseof skill in the art, for example, as described in U.S. Pat. No.4,554,101, incorporated herein by reference, which teaches theidentification and preparation of epitopes from amino acid sequences onthe basis of hydrophilicity. Moreover, numerous computer programs areavailable for use in predicting antigenic portions of proteins (seee.g., Jameson and Wolf, 1988; Wolf et al., 1988). Computerized peptidesequence analysis programs (e.g., DNAStar® software, DNAStar, Inc.,Madison, Wis.) may also be useful in designing synthetic peptides inaccordance with the present disclosure.

[0074] Syntheses of epitopic sequences, or peptides which include anantigenic epitope within their sequence, are readily achieved usingconventional synthetic techniques such as the solid phase method (e.g.,through the use of commercially available peptide synthesizer such as anApplied Biosystems Model 430A Peptide Synthesizer). Peptide antigenssynthesized in this manner may then be aliquotted in predeterminedamounts and stored in conventional manners, such as in aqueous solutionsor, even more preferably, in a powder or lyophilized state pending use.

[0075] In general, due to the relative stability of peptides, they maybe readily stored in aqueous solutions for fairly long periods of timeif desired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

2.6 Nucleic Acid Segments Encoding Crystal Protein Chimeras

[0076] The present invention also concerns DNA segments, both native,synthetic, and mutagenized, that can be synthesized, or isolated fromvirtually any source, that are free from total genomic DNA and thatencode the novel chimeric peptides disclosed herein. DNA segmentsencoding these peptide species may prove to encode proteins,polypeptides, subunits, functional domains, and the like of crystalprotein-related or other non-related gene products. In addition theseDNA segments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

[0077] As used herein, the term “DNA segment” refers to a DNA moleculethat has been isolated free of total genomic DNA of a particularspecies. Therefore, a DNA segment encoding a crystal protein or peptiderefers to a DNA segment that contains crystal protein coding sequencesyet is isolated away from, or purified free from, total genomic DNA ofthe species from which the DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species of Bacillus known as B. thuringiensis.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

[0078] Similarly, a DNA segment comprising an isolated or purifiedcrystal protein-encoding gene refers to a DNA segment which may includein addition to peptide encoding sequences, certain other elements suchas, regulatory sequences, isolated substantially away from othernaturally occurring genes or protein-encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein-, polypeptide- or peptide-encoding unit. As will be understoodby those in the art, this functional term includes both genomicsequences, operon sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides orpeptides.

[0079] “Isolated substantially away from other coding sequences” meansthat the gene of interest, in this case, a gene encoding a bacterialcrystal protein, forms the significant part of the coding region of theDNA segment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

[0080] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences that encodea Cry peptide species that includes within its amino acid sequence anamino acid sequence essentially as set forth in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:34.

[0081] The term “a sequence essentially as set forth in SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, orSEQ ID NO:34” means that the sequence substantially corresponds to aportion of the sequence of either SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34 and hasrelatively few amino acids that are not identical to, or a biologicallyfunctional equivalent of, the amino acids of any of these sequences. Theterm “biologically functional equivalent” is well understood in the artand is further defined in detail herein (e.g., see IllustrativeEmbodiments). Accordingly, sequences that have between about 70% andabout 80%, or more preferably between about 81% and about 90%, or evenmore preferably between about 91% and about 99% amino acid sequenceidentity or fuictional equivalence to the amino acids of SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, orSEQ ID NO:34 will be sequences that are “essentially as set forth in SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, or SEQ ID NO:34.”

[0082] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding regionor may include various internal sequences, i.e., introns, which areknown to occur within genes.

[0083] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding either of the peptidesequences disclosed in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, or that areidentical to or complementary to DNA sequences which encode any of thepeptides disclosed in SEQ ID NO:10, SEQ ID NO:12 SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, and particularlythose DNA segments disclosed in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. For example,DNA sequences such as about 14 nucleotides, and that are up to about10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500,about 200, about 100, about 50, and about 14 base pairs in length(including all intermediate lengths) are also contemplated to be useful.

[0084] It will be readily understood that “intermediate lengths”, inthese contexts, means any length between the quoted ranges, such as 14,15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50,51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; and up to and including sequences of about10,000 nucleotides and the like.

[0085] It will also be understood that this invention is not limited tothe particular nucleic acid sequences which encode peptides of thepresent invention, or which encode the amino acid sequences of SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, or SEQ ID NO:34, including those DNA sequences which areparticularly disclosed in SEQ ID NO:9, SEQ ID NO:11 SEQ ID NO:13, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. Recombinant vectorsand isolated DNA segments may therefore variously include thepeptide-coding regions themselves, coding regions bearing selectedalterations or modifications in the basic coding region, or they mayencode larger polypeptides that nevertheless include thesepeptide-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

[0086] The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

[0087] If desired, one may also prepare fusion proteins and peptides,e.g., where the peptide-coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins that may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0088] Recombinant vectors form further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment, whether encodinga full length protein or smaller peptide, is positioned under thecontrol of a promoter. The promoter may be in the form of the promoterthat is naturally associated with a gene encoding peptides of thepresent invention, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding segment or exon, for example,using recombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.7 Recombinant Vectors and Protein Expression

[0089] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a DNA segment encoding a crystalprotein or peptide in its natural environment. Such promoters mayinclude promoters normally associated with other genes, and/or promotersisolated from any bacterial, viral, eukaryotic, or plant cell.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, organism, oreven animal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al., 1989.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides. Appropriate promotersystems contemplated for use in high-level expression include, but arenot limited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

[0090] In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise contiguous aminoacid sequences from SEQ ID NO:10, SEQ ID NO:12 SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34; or any peptideepitope encoded by the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:33.

[0091] Methods for the recombinant expression of crystal proteins andvectors useful in the expression of DNA constructs encoding crystalproteins are described in Intl. Pat. Appl. Publ. No. WO 95/02058,specifically incorporated herein by reference.

2.8 Recombinant Host Cells

[0092] TABLE 1 STRAINS DEPOSITED WITH NRRL STRAIN PLASMID ACCESSIONNUMBER DEPOSIT DATE EG11063 pEG1068 B-21579 Jun. 26, 1996 EG11074pEG1077 B-21580 Jun. 26, 1996 EG11091 pEG1092 B-21780 May 21, 1997EG11092 pEG1093 B-21635 Nov. 14, 1996 EG11735 pEG365 B-21581 Jun. 26,1996 EG11751 pEG378 B-21636 Nov. 14, 1996 EG11768 pEG381 B-21781 May 21,1997

[0093] These bacterial strains have been deposited with the AgriculturalResearch Culture Collection (NRRL), which is located at the followingaddress:

[0094] 1815 N. University Street

[0095] Peoria, Ill. 91904

[0096] U.S.A.

2.9 DNA Segments as Hybridization Probes and Primers

[0097] In addition to their use in directing the expression of crystalproteins or peptides of the present invention, the nucleic acidsequences contemplated herein also have a variety of other uses. Forexample, they also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsegments that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or SEQ ID NO:33 will find particular utility. Also, nucleic acidsegments which encode at least a 6 amino acid contiguous sequence fromSEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, or SEQ ID NO:34, are also preferred. Longer contiguousidentical or complementary sequences, e.g., those of about 20, 30, 40,50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (including allintermediate lengths and up to and including full-length sequences willalso be of use in certain embodiments.

[0098] The ability of such nucleic acid probes to specifically hybridizeto crystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

[0099] Nucleic acid molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so, identical or complementary to DNA sequencesof SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27,SEQ ID NO:29, or SEQ ID NO:33, are particularly contemplated ashybridization probes for use in, e.g., Southern and Northern blotting.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 10-14 and about 100 or 200 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

[0100] Of course, fragments may also be obtained by other techniquessuch as, e.g., by mechanical shearing or by restriction enzymedigestion. Small nucleic acid segments or fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.Nos. 4,683,195 and 4,683,202 (each specifically incorporated herein byreference), by introducing selected sequences into recombinant vectorsfor recombinant production, and by other recombinant DNA techniquesgenerally known to those of skill in the art of molecular biology.

[0101] Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. Such selectiveconditions tolerate little, if any, mismatch between the probe and thetemplate or target strand, and would be particularly suitable forisolating crystal protein-encoding DNA segments. Detection of DNAsegments via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (eachspecifically incorporated herein by reference) are exemplary of themethods of hybridization analyses. Teachings such as those found in thetexts of Maloy et al., 1994; Segal 1976; Prokop, 1991; and Kuby, 1994,are particularly relevant.

[0102] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0103] In certain embodiments, it will be advantageous to employ nucleicacid sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as urease, alkaline phosphatase orperoxidase, instead of radioactive or other environmental undesirablereagents. In the case of enzyme tags, colorimetric indicator substratesare known that can be employed to provide a means visible to the humaneye or spectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

[0104] In general, it is envisioned that the hybridization probesdescribed herein will be useful both as reagents in solutionhybridization as well as in embodiments employing a solid phase. Inembodiments involving a solid phase, the test DNA (or RNA) is adsorbedor otherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.10 Biological Functional Equivalents

[0105] Modification and changes may be made in the structure of thepeptides of the present invention and DNA segments which encode them andstill obtain a functional molecule that encodes a protein or peptidewith desirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein and/orincreasing the insect-host range. These improvements may also beaccomplished by modifying the sequence of the protein or DNA to increasethe expression of the recombinant transgene in a plant cell. The aminoacid changes may be achieved by changing the codons of the DNA sequence,according to the codons given in Table 2. TABLE 2 Amino Acid CodonsAlanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp DGAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU GlycineGly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUCAUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0106] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated by theinventors that various changes may be made in the peptide sequences ofthe disclosed compositions, or corresponding DNA sequences which encodesaid peptides without appreciable loss of their biological utility oractivity.

[0107] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0108] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0109] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0110] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

[0111] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0112] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those which arewithin ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred.

[0113] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

2.11 Site-Specific Mutagenesis

[0114] Site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, for example, incorporating one ormore of the foregoing considerations, by introducing one or morenucleotide sequence changes into the DNA. Site-specific mutagenesisallows the production of mutants through the use of specificoligonucleotide sequences which encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 17 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues on both sides of the junctionof the sequence being altered.

[0115] In general, the technique of site-specific mutagenesis is wellknown in the art, as exemplified by various publications. As will beappreciated, the technique typically employs a phage vector which existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage are readily commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis whicheliminates the step of transferring the gene of interest from a plasmidto a phage.

[0116] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0117] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis isprovided as a means of producing potentially useful species and is notmeant to be limiting as there are other ways in which sequence variantsof peptides and the DNA sequences encoding them may be obtained. Forexample, recombinant vectors encoding the desired peptide sequence maybe treated with mutagenic agents, such as hydroxylamine, to obtainsequence variants.

2.12 Crystal Protein Compositions as Insecticides and Methods of use

[0118] The inventors contemplate that the chimeric crystal proteincompositions disclosed herein will find particular utility asinsecticides for topical and/or systemic application to field crops,grasses, fruits and vegetables, and ornamental plants. In a preferredembodiment, the bioinsecticide composition comprises an oil flowablesuspension of bacterial cells which expresses a novel crystal proteindisclosed herein. Preferably the cells are B. thuringiensis cells,however, any such bacterial host cell expressing the novel nucleic acidsegments disclosed herein and producing a crystal protein iscontemplated to be useful, such as B. megaterium, B. subtilis, E. coli,or Pseudomonas spp.

[0119] In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells are B. thuringiensis cells, however, bacteriasuch as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful.

[0120] In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or collodial concentrate.This powder comprises bacterial cells which expresses a novel crystalprotein disclosed herein. Preferred bacterial cells are B. thuringiensiscells, however, bacteria such as B. megaterium, B. subtilis, E. coli, orPseudomonas spp. cells transformed with a DNA segment disclosed hereinand expressing the crystal protein are also contemplated to be useful.Such dry forms of the insecticidal compositions may be formulated todissolve immediately upon wetting, or alternatively, dissolve in acontrolled-release, sustained-release, or other time-dependent manner.

[0121] In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the crystal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

[0122] For these methods involving application of bacterial cells, thecellular host containing the crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

[0123] When the insecticidal compositions comprise intact B.thuringiensis cells expressing the protein of interest, such bacteriamay be formulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include Theologicalagents, surfactants, emulsifiers, dispersants, or polymers.

[0124] Alternatively, the novel chimeric Cry proteins may be prepared byrecombinant bacterial expression systems in vitro and isolated forsubsequent field application. Such protein may be either in crude celllysates, suspensions, colloids, etc., or alternatively may be purified,refined, buffered, and/or further processed, before formulating in anactive biocidal formulation. Likewise, under certain circumstances, itmay be desirable to isolate crystals and/or spores from bacterialcultures expressing the crystal protein and apply solutions,suspensions, or collodial preparations of such crystals and/or spores asthe active bioinsecticidal composition.

[0125] Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

[0126] The insecticide compositions described may be made by formulatingeither the bacterial cell, crystal and/or spore suspension, or isolatedprotein component with the desired agriculturally-acceptable carrier.The compositions may be formulated prior to administration in anappropriate means such as lyophilized, freeze-dried, dessicated, or inan aqueous carrier, medium or suitable diluent, such as saline or otherbuffer. The formulated compositions may be in the form of a dust orgranular material, or a suspension in oil (vegetable or mineral), orwater or oil/water emulsions, or as a wettable powder, or in combinationwith any other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, e.g., inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in insecticide formulationtechnology; these are well known to those skilled in insecticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the insecticidal composition withsuitable adjuvants using conventional formulation techniques.

[0127] The insecticidal compositions of this invention are applied tothe environment of the target coleopteran insect, typically onto thefoliage of the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

[0128] Other application techniques, e.g., dusting, sprinkling, soaking,soil injection, seed coating, seedling coating, spraying, aerating,misting, atomizing, and the like, are also feasible and may be requiredunder certain circumstances such as e.g., insects that cause root orstalk infestation, or for application to delicate vegetation orornamental plants. These application procedures are also well-known tothose of skill in the art.

[0129] The insecticidal composition of the invention may be employed inthe method of the invention singly or in combination with othercompounds, including and not limited to other pesticides. The method ofthe invention may also be used in conjunction with other treatments suchas surfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

[0130] The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 0.5% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 0.5% to about 99% or more by weightof the composition, while liquid formulations may generally comprisefrom about 0.5% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10¹² cells/mg.

[0131] The insecticidal formulation may be administered to a particularplant or target area in one or more applications as needed, with atypical field application rate per hectare ranging on the order of fromabout 50 g to about 500 g of active ingredient, or of from about 500 gto about 1000 g, or of from about 1000 g to about 5000 g or more ofactive ingredient.

2.13 Antibody Compositions and Methods for Producing

[0132] In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the crystalproteins disclosed herein. Means for preparing and characterizingantibodies are well known in the art (See, e.g., Harlow and Lane, 1988;incorporated herein by reference). The methods for generating monoclonalantibodies (mAbs) generally begin along the same lines as those forpreparing polyclonal antibodies. Briefly, a polyclonal antibody isprepared by immunizing an animal with an immunogenic composition inaccordance with the present invention and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits, a rabbitis a preferred choice for production of polyclonal antibodies.

[0133] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0134] As is also well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0135] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster, injection mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs.

[0136] mAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265(specifically incorporated herein by reference). Typically, thistechnique involves immunizing a suitable animal with a selectedimmunogen composition, e.g., a purified or partially purified crystalprotein, polypeptide or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

[0137] Following immunization, somatic cells with the potential forproducing antibodies, specifically B lymphocytes (B cells), are selectedfor use in the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0138] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0139] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

[0140] One preferred murine myeloma cell is the NS-1 myeloma cell line(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

[0141] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 ratio, though the ratio may vary from about20:1 to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (vol./vol.) PEG, (Gefter et al., 1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

[0142] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0143] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B-cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B-cells.

[0144] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0145] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

3. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0147]FIG. 1. The wild-type δ-endotoxins and the relevant restrictionsites that were used to construct the hybrid δ-endotoxins pertinent tothe invention are diagrammed in FIG. 1A. Only the DNA encoding theδ-endotoxin that is contained on the indicated plasmid (identified bythe “pEG” prefix) is shown. The B. thuringiensis strains containing theindicated plasmids are identified by the “EG” prefix. The hybridδ-endotoxins described in the invention are diagrammed in FIG. 1B andare aligned with the wild-type δ-endotoxins in FIG. 1A.

[0148]FIG. 2. An equal amount of each washed sporulated B. thuringiensisculture was analyzed by SDS-PAGE. Lane a: control Cry1Ac producing B.thuringiensis strain EG11070, b: EG11060, c: EG11062, d: EG11063, e:EG11065, f: EG11067, g: EG11071, h: EG11073, i: EG11074, j: EG11088, k:EG11090, and l: EG11091.

[0149]FIG. 3. Solubilized hybrid δ-endotoxins were exposed to trypsinfor 0, 15, 30, 60, and 120 minutes. The resulting material was analyzedby SDS-PAGE. The amount of active δ-endotoxin fragment remaining wasquantitated by scanning densitometry using a Molecular Dynamics model300A densitometer. The percent active toxin remaining was plotted versustime. Wild-type Cry1Ac δ-endotoxin (open box) served as the control.

[0150]FIG. 4. Schematic diagrams of the wild-type toxins and therelevant restriction sites that were used to construct the hybridδ-endotoxin encoded by pEG381 and expressed in EG11768. Only the DNAencoding the δ-endotoxin that is contained on the indicated plasmid(identified by the “pEG” prefix) is shown.

[0151]FIG. 5. Scematic diagram of the hybrid Bt toxin proteins. Thedifferent protein domains from Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Fa areindicated by different shading. The crystal formation of each of thesehybrid proteins is also indicated.

4. BRIEF DESCRIPTION OF THE SEQUENCES

[0152] SEQ ID NO:1 is oligonucleotide primer A.

[0153] SEQ ID NO:2 is oligonucleotide primer B.

[0154] SEQ ID NO:3 is oligonucleotide primer C.

[0155] SEQ ID NO:4 is oligonucleotide primer D.

[0156] SEQ ID NO:5 is oligonucleotide primer E.

[0157] SEQ ID NO:6 is oligonucleotide primer F.

[0158] SEQ ID NO:7 is oligonucleotide primer G.

[0159] SEQ ID NO:8 is oligonucleotide primer H.

[0160] SEQ ID NO:9 is the nucleotide and deduced amino acid sequences ofthe EG11063 hybrid δ-endotoxin.

[0161] SEQ ID NO:10 denotes in the three-letter abbreviation form, theamino acid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:9.

[0162] SEQ ID NO:11 is the nucleotide and deduced amino acid sequencesof the EG11074 hybrid δ-endotoxin.

[0163] SEQ ID NO:12 denotes in the three-letter abbreviation form, theamino acid sequence for the hybrid δ-endotoxin specified in SEQ IDNO:11.

[0164] SEQ ID NO:13 is the nucleotide and deduced amino acid sequencesof the EG11735 hybrid δ-endotoxin.

[0165] SEQ ID NO:14 denotes in the three-letter abbreviation form, theamino acid sequence for the hybrid δ-endotoxin specified in SEQ IDNO:13.

[0166] SEQ ID NO:15 is the 5′ exchange site for pEG1065, pEG1070, andpEG1074.

[0167] SEQ ID NO:16 is the 5′ exchange site for pEG1067, pEG1072, andpEG1076.

[0168] SEQ ID NO:17 is the 5′ exchange site for pEG1068, pEG1077, andpEG365.

[0169] SEQ ID NO:18 is the 5′ exchange site for pEG1088 and pEG1092.

[0170] SEQ ID NO:19 is the 5′ exchange site for pEG1089 and the 3′exchange site for pEG1070 and pEG1072.

[0171] SEQ ID NO:20 is the 5′ exchange site for pEG1091.

[0172] SEQ ID NO:21 is the 3′ exchange site for pEG1065, pEG1067,pEG1068, pEG1093, pEG378, and pEG 365.

[0173] SEQ ID NO:22 is the 3′ exchange site for pEG1088.

[0174] SEQ ID NO:23 is oligonucleotide Primer I.

[0175] SEQ ID NO:24 is oligonucleotide Primer J.

[0176] SEQ ID NO:25 is the nucleic acid sequence and deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11092.

[0177] SEQ ID NO:26 is the three-letter abbreviation form of the aminoacid sequence of the hybrid crystal protein produced by strain EG11092encoded by SEQ ID NO:25.

[0178] SEQ ID NO:27 is the nucleic acid sequence and the deduced aminoacid sequence of the hybrid crystal protein-encoding gene of EG11751.

[0179] SEQ ID NO:28 is the three-letter abbreviation form of the aminoacid sequence of the hybrid crystal protein produced by strain EG11751encoded by SEQ ID NO:27.

[0180] SEQ ID NO:29 is the nucleic acid sequence and the deduced aminoacid sequence of the hybrid crystal protein-encoding gene of EG11091.

[0181] SEQ ID NO:30 is the three-letter abbreviation form of the aminoacid sequence of the hybrid crystal protein produced by strain EG11091encoded by SEQ ID NO:29.

[0182] SEQ ID NO:31 is oligonucleotide primer K.

[0183] SEQ ID NO:32 is the 5′ exchange site for pEG378 and pEG381.

[0184] SEQ ID NO:33 is the nucleic acid sequence and the deduced aminoacid sequence of the hybrid crystal protein-encoding gene of EG11768.

[0185] SEQ ID NO:34 denotes in the three-letter abbreviation form, theamino acid sequence of the hybrid crystal protein produced by strain.EG11768 encoded by SEQ ID NO:33.

[0186] SEQ ID NO:35 is the 3′ exchange site for pEG1074, pEG1076,pEG1077 and pEG381.

5. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 5.1 Methods for Culturing B.thuringiensis to Produce Cry Proteins

[0187] The B. thuringiensis strains described herein may be culturedusing standard known media and fermentation techniques. Upon completionof the fermentation cycle, the bacteria may be harvested by firstseparating the B. thuringiensis spores and crystals from thefermentation broth by means well known in the art. The recovered B.thuringiensis spores and crystals can be formulated into a wettablepowder, a liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers and othercomponents to facilitate handling and application for particular targetpests. The formulation and application procedures are all well known inthe art and are used with commercial strains of B. thuringiensis (HD-1)active against Lepidoptera, e.g., caterpillars.

5.2 Recombinant Host Cells for Expression of Cry Genes

[0188] The nucleotide sequences of the subject invention can beintroduced into a wide variety of microbial hosts. Expression of thetoxin gene results, directly or indirectly, in the intracellularproduction and maintenance of the pesticide. With suitable hosts, e.g.,Pseudomonas, the microbes can be applied to the sites of lepidopteraninsects where they will proliferate and be ingested by the insects. Theresults is a control of the unwanted insects. Alternatively, the microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin produced in the cell. The treated cell then can beapplied to the environment of target pest(s). The resulting productretains the toxicity of the B. thuringiensis toxin.

[0189] Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility or toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and Gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

[0190] Characteristics of particular interest in selecting a host cellfor purposes of production include ease of introducing the B.thuringiensis gene into the host, availability of expression systems,efficiency of expression, stability of the pesticide in the host, andthe presence of auxiliary genetic capabilities. Characteristics ofinterest for use as a pesticide microcapsule include protectivequalities for the pesticide, such as thick cell walls, pigmentation, andintracellular packaging or formation of inclusion bodies; leaf affinity;lack of mammalian toxicity; attractiveness to pests for ingestion; easeof killing and fixing without damage to the toxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

[0191] Host organisms of particular interest include yeast, such asRhodotorula sp., Aureobasidium sp., Saccharomyces sp., andSporobolomyces sp.; phylloplane organisms such as Pseudomonas sp.,Erwinia sp. and Flavobacterium sp.; or such other organisms asEscherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and thelike. Specific organisms include Pseudomonas aeruginosa, P. fluorescens,Saccharomyces cerevisiae, B. thuringiensis, B. subtilis, E. coli,Streptomyces lividans and the like.

[0192] Treatment of the microbial cell, e.g., a microbe containing theB. thuringiensis toxin gene, can be by chemical or physical means, or bya combination of chemical and/or physical means, so long as thetechnique does not deleteriously affect the properties of the toxin, nordiminish the cellular capability in protecting the toxin. Examples ofchemical reagents are halogenating agents, particularly halogens ofatomic no. 17-80. More particularly, iodine can be used under mildconditions and for sufficient time to achieve the desired results. Othersuitable techniques include treatment with aldehydes, such asformaldehyde and glutaraldehye; anti-infectives, such as zephiranchloride and cetylpyridinium chloride; alcohols, such as isopropyl andethanol; various histologic fixatives, such as Lugol's iodine, Bouin'sfixative, and Helly's fixatives, (see e.g., Humason, 1967); or acombination of physical (heat) and chemical agents that preserve andprolong the activity of the toxin produced in the cell when the cell isadministered to a suitable host. Examples of physical means are shortwavelength radiation such as γ-radiation and X-radiation, freezing, UVirradiation, lyophilization, and the like. The cells employed willusually be intact and be substantially in the proliferative form whentreated, rather than in a spore form, although in some instances sporesmay be employed.

[0193] Where the B. thuringiensis toxin gene is introduced via asuitable vector into a microbial host, and said host is applied to theenvironment in a living state, it is essential that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment (crop and other insect habitats) with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

[0194] A large number of microorganisms are known to inhabit thephylloplane (the surface of the plant leaves) and/or the rhizosphere(the soil surrounding plant roots) of a wide variety of important crops.These microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus,Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus, andAzotobacter vinlandii; and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans.

5.3 Definitions

[0195] The following words and phrases have the meanings set forthbelow.

[0196] Broad-Spectrum: refers to a wide range of insect species.

[0197] Broad-Spectrum Insecticidal Activity (or broad insecthost-range): insecticidal activity exhibited by the presently disclosedhybrid crystal proteins against a wider range of insect species,relative to the non-hybrid proteins from which they were engineered(i.e. broader insect host-range).

[0198] Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

[0199] Insecticidal Activity: toxicity towards insects.

[0200] Insecticidal Specificity: the level of insecticidal activity of acrystal protein against a particular insect species. The presentlydisclosed hybrid proteins typically exhibit an increased insecticidalspecificity, relative to the non-hybrid proteins from which they wereengineered (i.e. a lower LC50).

[0201] Intraorder Specificity: the insecticidal activity of a particularcrystal protein towards insect species within an Order of insects (e.g.,Order Lepidoptera).

[0202] Interorder Specificity: the insecticidal activity of a particularcrystal protein towards insect species of different Orders (e.g., OrdersLepidoptera and Diptera).

[0203] LC₅₀: the lethal concentration of crystal protein that causes 50%mortality of the insects treated.

[0204] LC₉₅: the lethal concentration of crystal protein that causes 95%mortality of the insects treated.

[0205] Promoter: A recognition site on a DNA sequence or group of DNAsequences that provide an expression control element for a structuralgene and to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene.

[0206] Regeneration: The process of growing a plant from a plant cell(e.g., plant protoplast or explant).

[0207] Structural Gene: A gene that is expressed to produce apolypeptide.

[0208] Transformation: A process of introducing an exogenous DNAsequence (e.g., a vector, a recombinant DNA molecule) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

[0209] Transformed Cell: A cell whose DNA has been altered by theintroduction of an exogenous DNA molecule into that cell.

[0210] Transgene: An exogenous gene which when introduced into thegenome of a host cell through a process such as transformation,electroporation, particle bombardment, and the like, is expressed by thehost cell and integrated into the cells genome such that the trait ortraits produced by the expression of the transgene is inherited by theprogeny of the transformed cell.

[0211] Transgenic Cell: Any cell derived or regenerated from atransformed cell or derived from a transgenic cell. Exemplary transgeniccells include plant calli derived from a transformed plant cell andparticular cells such as leaf, root, stem, e.g., somatic cells, orreproductive (germ) cells obtained from a transgenic plant.

[0212] Transgenic Plant: A plant or progeny thereof derived from atransformed plant cell or protoplast, wherein the plant DNA contains anintroduced exogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

[0213] Vector: A DNA molecule capable of replication in a host celland/or to which another DNA segment can be operatively linked so as tobring about replication of the attached segment. A plasmid is anexemplary vector.

5.4 Probes and Primers

[0214] In another aspect, DNA sequence information provided by theinvention allows for the preparation of relatively short DNA (or RNA)sequences having the ability to specifically hybridize to gene sequencesof the selected polynucleotides disclosed herein. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein gene sequence, e.g., asequence such as that shown in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. The abilityof such nucleic acid probes to specifically hybridize to a crystalprotein-encoding gene sequence lends them particular utility in avariety of embodiments. Most importantly, the probes may be used in avariety of assays for detecting the presence of complementary sequencesin a given sample.

[0215] In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCR™ using such primers.

[0216] To provide certain of the advantages in accordance with thepresent invention, a preferred nucleic acid sequence employed forhybridization studies or assays includes sequences that arecomplementary to at least a 14 to 30 or so long nucleotide stretch of acrystal protein-encoding sequence, such as that shown in SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, orSEQ ID NO:33. A size of at least 14 nucleotides in length helps toensure that the fragment will be of sufficient length to form a duplexmolecule that is both stable and selective. Molecules havingcomplementary sequences over stretches greater than 14 bases in lengthare generally preferred, though, in order to increase stability andselectivity of the hybrid, and thereby improve the quality and degree ofspecific hybrid molecules obtained. One will generally prefer to designnucleic acid molecules having gene-complementary stretches of 14 to 20nucleotides, or even longer where desired. Such fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, by application of nucleic acid reproduction technology, such asthe PCR™ technology of U.S. Pat. Nos. 4,683,195, and 4,683,202 (eachspecifically incorporated herein by reference), or by excising selectedDNA fragments from recombinant plasmids containing appropriate insertsand suitable restriction sites.

5.5 Expression Vectors

[0217] The present invention contemplates an expression vectorcomprising a polynucleotide of the present invention. Thus, in oneembodiment an expression vector is an isolated and purified DNA moleculecomprising a promoter operatively linked to an coding region thatencodes a polypeptide of the present invention, which coding region isoperatively linked to a transcription-terminating region, whereby thepromoter drives the transcription of the coding region.

[0218] As used herein, the term “operatively linked” means that apromoter is connected to an coding region in such a way that thetranscription of that coding region is controlled and regulated by thatpromoter. Means for operatively linking a promoter to a coding regionare well known in the art.

[0219] Promoters that function in bacteria are well known in the art.Exemplary and preferred promoters for the Bacillus crystal proteinsinclude the sigA, sigE, and sigK gene promoters. Alternatively, thenative, mutagenized, or recombinant crystal protein-encoding genepromoters themselves can be used.

[0220] Where an expression vector of the present invention is to be usedto transform a plant, a promoter is selected that has the ability todrive expression in plants. Promoters that function in plants are alsowell known in the art. Useful in expressing the polypeptide in plantsare promoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

[0221] A promoter is also selected for its ability to direct thetransformed plant cell's or transgenic plant's transcriptional activityto the coding region. Structural genes can be driven by a variety ofpromoters in plant tissues. Promoters can be near-constitutive, such asthe CaMV 35S promoter, or tissue-specific or developmentally specificpromoters affecting dicots or monocots.

[0222] Where the promoter is a near-constitutive promoter such as CaMV35S, increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

[0223] An exemplary tissue-specific promoter is the lectin promoter,which is specific for seed tissue. The Lectin protein in soybean seedsis encoded by a single gene (Le1) that is only expressed during seedmaturation and accounts for about 2 to about 5% of total seed mRNA. Thelectin gene and seed-specific promoter have been fully characterized andused to direct seed specific expression in transgenic tobacco plants(Vodkin et al., 1983; Lindstrom et al., 1990.)

[0224] An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

[0225] A transgenic plant of the present invention produced from a plantcell transformed with a tissue specific promoter can be crossed with asecond transgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

[0226] Exemplary tissue-specific promoters are corn sucrose synthetase 1(Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989),corn light harvesting complex (Simpson, 1986), corn heat shock protein(Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen etal., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase(Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge etal., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), beanglycine rich protein 1 (Keller et al., 1989), CaMV 35s transcript (Odellet al., 1985) and Potato patatin (Wenzler et al., 1989). Preferredpromoters are the cauliflower mosaic virus (CaMV 35S) promoter and theS-E9 small subunit RuBP carboxylase promoter.

[0227] The choice of which expression vector and ultimately to whichpromoter a polypeptide coding region is operatively linked dependsdirectly on the functional properties desired, e.g., the location andtiming of protein expression, and the host cell to be transformed. Theseare well known limitations inherent in the art of constructingrecombinant DNA molecules. However, a vector useful in practicing thepresent invention is capable of directing the expression of thepolypeptide coding region to which it is operatively linked.

[0228] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described(Rogers et al, 1987). However, several other plant integrating vectorsystems are known to function in plants including pCaMVCN transfercontrol vector described (Fromm et al, 1985). pCaMVCN (available fromPharmacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV35S promoter.

[0229] In preferred embodiments, the vector used to express thepolypeptide includes a selection marker that is effective in a plantcell, preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al, 1988).

[0230] RNA polymerase transcribes a coding DNA sequence through a sitewhere polyadenylation occurs. Typically, DNA sequences located a fewhundred base pairs downstream of the polyadenylation site serve toterminate transcription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

[0231] Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011 (each of which is specificallyincorporated herein by reference). Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

[0232] A variety of methods has been developed to operatively link DNAto vectors via complementary cohesive termini or blunt ends. Forinstance, complementary homopolymer tracts can be added to the DNAsegment to be inserted and to the vector DNA. The vector and DNA segmentare then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

[0233] A coding region that encodes a polypeptide having the ability toconfer insecticidal activity to a cell is preferably a chimeric B.thuringiensis crystal protein-encoding gene. In preferred embodiments,such a polypeptide has the amino acid residue sequence of SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, orSEQ ID NO:34; or a functional equivalent of one or more of thosesequences. In accordance with such embodiments, a coding regioncomprising the DNA sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33 is alsopreferred.

5.6 Transformed or Transgenic Plant Cells

[0234] A bacterium, a yeast cell, or a plant cell or a plant transformedwith an expression vector of the present invention is also contemplated.A transgenic bacterium, yeast cell, plant cell or plant derived fromsuch a transformed or transgenic cell is also contemplated. Means fortransforming bacteria and yeast cells are well known in the art.Typically, means of transformation are similar to those well known meansused to transform other bacteria or yeast such as E. coli or S.cerevisiae.

[0235] Methods for DNA transformation of plant cells includeAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. Each of these methodshas distinct advantages and disadvantages. Thus, one particular methodof introducing genes into a particular plant strain may not necessarilybe the most effective for another plant strain, but it is well knownwhich methods are useful for a particular plant strain.

[0236] There are many methods for introducing transforming DNA segmentsinto cells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as infection by A. tumefaciensand related Agrobacterium, direct delivery of DNA such as, for example,by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993),by desiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

[0237] Technology for introduction of DNA into cells is well-known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, 1973); (2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viralvectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a;1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;Wagner et al., 1992).

5.6.1 Electroporation

[0238] The application of brief, high-voltage electric pulses to avariety of animal and plant cells leads to the formation ofnanometer-sized pores in the plasma membrane. DNA is taken directly intothe cell cytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of clones genes and for establishment of celllines that carry integrated copies of the gene of interest.Electroporation, in contrast to calcium phosphate-mediated transfectionand protoplast fusion, frequently gives rise to cell lines that carryone, or at most a few, integrated copies of the foreign DNA.

[0239] The introduction of DNA by means of electroporation, iswell-known to those of skill in the art. In this method, certain cellwall-degrading enzymes, such as pectin-degrading enzymes, are employedto render the target recipient cells more susceptible to transformationby electroporation than untreated cells. Alternatively, recipient cellsare made more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

5.6.2 Microprojectile Bombardment

[0240] A further advantageous method for delivering transforming DNAsegments to plant cells is microprojectile bombardment. In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

[0241] An advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly stably transforming monocots,is that neither the isolation of protoplasts (Cristou et al., 1988) northe susceptibility to Agrobacterium infection is required. Anillustrative embodiment of a method for delivering DNA into maize cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withcorn cells cultured in suspension. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing damage inflicted on the recipient cells by projectiles that aretoo large.

[0242] For the bombardment, cells in suspension are preferablyconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the macroprojectile stopping plate. If desired, one or morescreens are also positioned between the acceleration device and thecells to be bombarded. Through the use of techniques set forth hereinone may obtain up to 1000 or more foci of cells transiently expressing amarker gene. The number of cells in a focus which express the exogenousgene product 48 hours post-bombardment often range from 1 to 10 andaverage 1 to 3.

[0243] In bombardment transformation, one may optimize theprebombardment culturing conditions and the bombardment parameters toyield the maximum numbers of stable transformants. Both the physical andbiological parameters for bombardment are important in this technology.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmids. It is believed that pre-bombardment manipulationsare especially important for successful transformation of immatureembryos.

[0244] Accordingly, it is contemplated that one may wish to adjustvarious of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

[0245] The methods of particle-mediated transformation is well-known tothose of skill in the art. U.S. Pat. No. 5,015,580 (specificallyincorporated herein by reference) describes the transformation ofsoybeans using such a technique.

5.6.3 Agrobacterium-Mediated Transfer

[0246] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). The genetic engineering of cottonplants using Agrobacterium-mediated transfer is described in U.S. Pat.No. 5,004,863 (specifically incorporated herein by reference), while thetransformation of lettuce plants is described in U.S. Pat. No. 5,349,124(specifically incorporated herein by reference). Further, theintegration of the Ti-DNA is a relatively precise process resulting infew rearrangements. The region of DNA to be transferred is defined bythe border sequences, and intervening DNA is usually inserted into theplant genome as described (Spielmann et al., 1986; Jorgensen et al.,1987).

[0247] Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

[0248] Agrobacterium-mediated transformation of leaf disks and othertissues such as cotyledons and hypocotyls appears to be limited toplants that Agrobacterium naturally infects. Agrobacterium-mediatedtransformation is most efficient in dicotyledonous plants. Few monocotsappear to be natural hosts for Agrobacterium, although transgenic plantshave been produced in asparagus using Agrobacterium vectors as described(Bytebier et al., 1987). Therefore, commercially important cereal grainssuch as rice, corn, and wheat must usually be transformed usingalternative methods. However, as mentioned above, the transformation ofasparagus using Agrobacterium can also be achieved (see, e.g., Bytebieret al., 1987).

[0249] A transgenic plant formed using Agrobacterium transformationmethods typically contains a single gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedgene. However, inasmuch as use of the word “heterozygous” usuallyimplies the presence of a complementary gene at the same locus of thesecond chromosome of a pair of chromosomes, and there is no such gene ina plant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

[0250] More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants produced for enhanced carboxylase activity relativeto a control (native, non-transgenic) or an independent segreganttransgenic plant.

[0251] It is to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes thatencode a polypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

[0252] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

[0253] Application of these systems to different plant strains dependsupon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts are described (see, e.g., Fujimura et al., 1985; Toriyama etal., 1986; Yamada et al., 1986; Abdullah et al., 1986).

[0254] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,1988). In addition, “particle gun” or high-velocity microprojectiletechnology can be utilized (Vasil, 1992).

[0255] Using that latter technology, DNA is carried through the cellwall and into the cytoplasm on the surface of small metal particles asdescribed (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988).The metal particles penetrate through several layers of cells and thusallow the transformation of cells within tissue explants.

5.7 Production of Insect-Resistant Transgenic Plants

[0256] Thus, the amount of a gene coding for a polypeptide of interest(i.e., a bacterial crystal protein or polypeptide having insecticidalactivity against one or more insect species) can be increased in plantsuch as corn by transforming those plants using particle bombardmentmethods (Maddock et al., 1991). By way of example, an expression vectorcontaining a coding region for a B. thuringiensis crystal protein and anappropriate selectable marker is transformed into a suspension ofembryonic maize (corn) cells using a particle gun to deliver the DNAcoated on microprojectiles. Transgenic plants are regenerated fromtransformed embryonic calli that express the disclosed insecticidalcrystal proteins. Particle bombardment has been used to successfullytransform wheat (Vasil et al., 1992).

[0257] DNA can also be introduced into plants by direct DNA transferinto pollen as described (Zhou et al., 1983; Hess, 1987; Luo et al.,1988). Expression of polypeptide coding genes can be obtained byinjection of the DNA into reproductive organs of a plant as described(Pena et al., 1987). DNA can also be injected directly into the cells ofimmature embryos and the rehydration of desiccated embryos as described(Neuhaus et al., 1987; Benbrook et al., 1986).

[0258] The development or regeneration of plants from either singleplant protoplasts or various explants is well known in the art(Weissbach and Weissbach, 1988). This regeneration and growth processtypically includes the steps of selection of transformed cells,culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

[0259] The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

[0260] This procedure typically produces shoots within two to fourmonths and those shoots are then transferred to an appropriateroot-inducing medium containing the selective agent and an antibiotic toprevent bacterial growth. Shoots that rooted in the presence of theselective agent to form plantlets are then transplanted to soil or othermedia to allow the production of roots. These procedures vary dependingupon the particular plant strain employed, such variations being wellknown in the art.

[0261] Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

[0262] A transgenic plant of this invention thus has an increased amountof a coding region (e.g., a cry gene) that encodes one or more of theChimeric Cry polypeptides disclosed herein. A preferred transgenic plantis an independent segregant and can transmit that gene and its activityto its progeny. A more preferred transgenic plant is homozygous for thatgene, and transmits that gene to all of its offspring on sexual mating.Seed from a transgenic plant may be grown in the field or greenhouse,and resulting sexually mature transgenic plants are self-pollinated togenerate true breeding plants. The progeny from these plants become truebreeding lines that are evaluated for, by way of example, increasedinsecticidal capacity against Coleopteran insects, preferably in thefield, under a range of environmental conditions. The inventorscontemplate that the present invention will find particular utility inthe creation of transgenic corn, soybeans, cotton, wheat, oats, barley,other grains, vegetables, fruits, fruit trees, berries, turf grass,ornamentals, shrubs and trees.

6. EXAMPLES

[0263] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

6.1 Example 1 Construction of Hybrid B. thuringiensis δ-Endotoxins

[0264] The B. thuringiensis shuttle vectors pEG853, pEG854, and pEG857which are used in the present invention have been described (Baum etal., 1990). pEG857 contains the Cry1Ac gene cloned into pEG853 as anSphI-BamHI DNA fragment. pEG1064 was constructed in such a way that theKpnI site within the cry1Ac gene was preserved and the KpnI site in thepEG857 multiple cloning site (MCS) was eliminated. This was accomplishedby sequentially subjecting pEG857 DNA to limited KpnI digestion so thatonly one KpnI site is cut, filling in the KpnI 5′ overhang by Klenowfragment of DNA polymerase I to create blunt DNA ends, and joining theblunt ends of DNA by T4 DNA ligase. pEG318 contains the cry1F gene(Chambers et al., 1991) cloned into the XhoI site of pEG854 as anXhoI-SalI DNA fragment. pEG315 contains the cry1C gene from strainEG6346 (Chambers et al., 1991) cloned into the XhoI-BamHI sites ofpEG854 as a SalI-BamHI DNA fragment.

[0265]FIG. 1A shows a schematic representation of the DNA encoding thecomplete cry1Ac, cry1Ab, cry1C, and cry1F genes contained onpEG854/pEG1064, pEG20, pEG3 15, and pEG318, respectively. Uniquerestriction sites that were used in constructing certain hybrid genesare also shown. FIG. 1B shows a schematic representation of hybrid genespertaining to the present invention. In some cases standard PCR™amplification with mutagenic oligonucleotide primers were used toincorporate appropriate restrictions sites into DNA fragments used forhybrid gene construction. Certain hybrid gene constructions could not beaccomplished by restriction fragment subcloning. In those instances,PCR™ overlap extension (POE) was used to construct the desired hybridgene (Horton et al., 1989). The following oligonucleotide primers(purchased from Integrated DNA Technologies Inc., Coralville, Iowa) wereused: Primer A: 5′-GGATAGCACTCATCAAAGGTACC-3′ (SEQ ID NO: 1) Primer B:5′-GAAGATATCCAATTCGAACAGTTTCCC-3′ (SEQ ID NO: 2) Primer C:5′-CATATTCTGCCTCGAGTGTTGCAGTAAC-3′ (SEQ ID NO: 3) Primer D:5′-CCCGATCGGCCGCATGC-3′ (SEQ ID NO: 4) Primer E: 5′-CATTGGAGCTCTCCATG-3′(SEQ ID NO: 5) Primer F: 5′-GCACTACGATGTATCC-3′ (SEQ ID NO: 6) Primer G:5′-CATCGTAGTGCAACTCTTAC-3′ (SEQ ID NO: 7) Primer H:5′-CCAAGAAAATACTAGAGCTCTTGTTAAAAAAGGTGTTCC-3′ (SEQ ID NO: 8) Primer I:5′-ATTTGAGTAATACTATCC-3′ (SEQ ID NO: 23) Primer J:5′-ATTACTCAAATACCATTGG-3′ (SEQ ID NO: 24) Primer K:5′-TCGTTGCTCTGTTCCCG-3′ (SEQ ID NO: 31)

[0266] The plasmids described in FIG. 1B containing the hybridδ-endotoxin genes pertinent to this invention are described below.Isolation or purification of DNA fragments generated by restriction ofplasmid DNA, PCR™ amplification, or POE refers to the sequentialapplication of agarose-TAE gel electrophoresis and use of the GenecleanKit (Bio 101) following the manufacturer's recommendation. pEG1065 wasconstructed by PCR™ amplification of the cry1F DNA fragment using primerpair A and B and pEG318 as the DNA template. The resulting PCR™ productwas isolated, cut with AsuII and KpnI, and used to replace thecorresponding AsuII-KpnI DNA fragment in pEG857. Plasmid pEG1067 wasconstructed using POE and DNA fragments SauI-KpnI of cry1F andAsuII-ClaI of cry1Ac that were isolated from pEG318 and pEG857,respectively. The resulting POE product was PCR™ amplified with primerpair A and B, cut with AsuII and KpnI, and used to replace thecorresponding AsuII-KpnI fragment in pEG857.

[0267] pEG1068 was constructed by replacing the SacI-KpnI DNA fragmentof cry1Ac isolated from pEG857 with the corresponding SacI-KpnI DNAfragment isolated from cry1F (pEG318). pEG1070 was constructed byreplacing the SacI-KpnI DNA fragment isolated from pEG1065 with thecorresponding SacI-KpnI DNA fragment isolated from cry1Ac (pEG857).pEG1072 was constructed by replacing the SacI-KpnI DNA fragment isolatedfrom pEG1067 with the corresponding SacI-KpnI DNA fragment isolated fromcry1Ac (pEG857). pEG1074, pEG1076, and pEG1077 were constructed byreplacing the SphI-XhoI DNA fragment from pEG1064 with the PCR™amplified SphI-XhoI DNA fragment from pEG1065, pEG1067, pEG1068,respectively, using primer pairs C and D. pEG1089 was constructed byreplacing the SphI-SacI DNA fragment of pEG1064 with the isolated andSphI and SacI cut PCR™ product of cry1F that was generated using primerpair D and E and the template pEG318.

[0268] pEG1091 was constructed by replacing the SphI-SacI DNA fragmentof pEG1064 with the isolated and SphI and SacI cut PCR™ product of cry1Cthat was generated using primer pair D and H and the template pEG315.

[0269] pEG1088 was constructed by POE using a cry1Ac DNA fragmentgenerated using primer pair B and F and a cry1C DNA fragment generatedusing primer pair A and G. The SacI-KpnI fragment was isolated from theresulting POE product and used to replace the corresponding SacI-KpnIfragment in pEG1064.

[0270] pEG365 was constructed by first replacing the SphI-KpnI DNAfragment from pEG1065 with the corresponding cry1Ab DNA fragmentisolated from pEG20 to give pEG364. The SacI-KpnI DNA fragment frompEG364 was then replaced with the corresponding cry1F DNA fragmentisolated from pEG318.

[0271] pEG1092 was constructed by replacing the KpnI-BamHI DNA fragmentfrom pEG1088 with the corresponding DNA fragment isolated from pEG315.pEG1092 is distinct from the cry1Ab/cry1C hybrid δ-endotoxin genedisclosed in Intl. Pat. Appl. Publ. No. WO 95/06730.

[0272] pEG1093 was constructed by replacing the SphI-AsuII DNA fragmentfrom pEG1068 with the corresponding SphI-AsuII DNA fragment isolatedfrom pEG20.

[0273] pEG378 was constructed by POE using a cry1Ac DNA fragmentgenerated using primer pair B and I using pEG857 as the template and acry1F DNA fragment generated using primer pair A and J using pEG318 asthe template. The resulting POE product was cut with AsuII and KpnI andthe resulting isolated DNA fragment used to replace the correspondingAsuII-KpnI DNA fragment in pEG1064.

[0274] pEG381 was constructed by replacing the AsuII-XhoI DNA fragmentin pEG1064 with the corresponding AsuII-XhoI DNA fragment isolated fromthe PCR™ amplification of pEG378 using primer pair C and K.

6.2 Example 2 Production of the Hybrid Toxins in B. thuringiensis

[0275] The plasmids encoding the hybrid toxins described in Example 1were transformed into B. thuringiensis as described (Mettus andMacaluso, 1990). The resulting B. thuringiensis strains were grown in 50ml of C-2 medium until the culture was fully sporulated and lysed(approximately 48 hr.). Since crystal formation is a prerequisite forefficient commercial production of δ-endotoxins in B. thuringiensis,microscopic analysis was used to identify crystals in the sporulatedcultures (Table 4). TABLE 3 CRYSTAL FORMATION BY THE HYBRID δ-ENDOTOXINSCrystal Strain Plasmid Parent δ-Endotoxins Formation EG11060 pEG1065Cry1Ac + Cry1F + EG11062 pEG1067 Cry1Ac + Cry1F + EG11063 pEG1068Cry1Ac + Cry1F + EG11065 pEG1070 Cry1Ac + Cry1F − EG11067 pEG1072Cry1Ac + Cry1F − EG11071 pEG1074 Cry1Ac + Cry1F + EG11073 pEG1076Cry1Ac + Cry1F + EG11074 pEG1077 Cry1Ac + Cry1F + EG11087 pEG1088Cry1Ac + Cry1C − EG11088 pEG1089  Cry1F + Cry1Ac − EG11090 pEG1091 Cry1C + Cry1Ac − EG11091 pEG1092 Cry1Ac + Cry1C + EG11092 pEG1093Cry1Ab + Cry1Ac + Cry1F + EG11735 pEG365 Cry1Ab + Cry1F + Cry1Ac +EG11751 pEG378 Cry1Ac + Cry1F + EG11768 pEG381 Cry1Ac + Cry1F +

[0276] The δ-endotoxin production for some of the B. thuringiensisstrains specified in Table 3 was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described byBaum et al., 1990. Equal volume cultures of each B. thuringiensis strainwere grown in C-2 medium until fully sporulated and lysed. The cultureswere centrifuged and the spore/crystal pellet was washed twice withequal volumes of distilled deionized water. The final pellet wassuspended in half the culture volume of 0.005% Triton X-100®. An equalvolume of each washed culture was analyzed by SDS-PAGE as shown in FIG.2.

[0277] The majority of hybrids involving Cry1Ac and Cry1F formed stablecrystals in B. thuringiensis A notable exception is EG11088 in which theactive toxin fragment would be the reciprocal exchange of EG11063. Twoof the three hybrids involving Cry1Ac and Cry1C, EG11087 and EG11090,failed to produce crystal in B. thuringiensis even though thesereciprocal hybrids mimic the activated toxin fragments ofcrystal-forming EG11063 and EG11074.

[0278] Every strain that was examined by SDS-PAGE produced some level ofδ-endotoxin. As expected, however, those cultures identified as crystalnegative produced very little protein (e.g., lane e: EG11065, lane f:EG11067, lane j: EG11088, and lane k: EG11090). For reference, typicalyields from a crystal forming δ-endotoxin is shown for Cry1Ac (lane a).Several hybrid δ-endotoxins produce comparable levels of proteinincluding EG11060 (lane b), EG11062 (lane c), EG11063 (lane d; SEQ IDNO:10), and EG11074 (lane i; SEQ ID NO:12). The data clearly show thatefficient hybrid δ-endotoxin production in B. thuringiensis isunpredictable and varies depending on the parent δ-endotoxins used toconstruct the hybrid.

6.3 Example 3 Proteolytic Processing of the Hybrid δ-Endotoxins

[0279] Proteolytic degradation of the protoxin form of the δ-endotoxinto a stable active toxin occurs once δ-endotoxin crystals aresolubilized in the larval midgut. One measure of the potential activityof δ-endotoxins is the stability of the active δ-endotoxin in aproteolytic environment. To test the proteolytic sensitivity of thehybrid δ-endotoxins, solubilized toxin was subjected to trypsindigestion. The δ-endotoxins were purified from sporulated B.thuringiensis cultures and quantified as described (Chambers et al.,1991). Exactly 250 μg of each hybrid δ-endotoxin crystal was solubilizedin 30 mM NaHCO₃, 10 mM DTT (total volume 0.5 ml). Trypsin was added tothe solubilized toxin at a 1:10 ratio. At appropriate time points 50 μlaliquots were removed to 50 μl Laemmli buffer, heated to 100° C. for 3min., and frozen in a dry-ice ethanol bath for subsequent analysis. Thetrypsin digests of the solubilized toxins were analyzed by SDS-PAGE andthe amount of active δ-endotoxin at each time point was quantified bydensitometry. A graphic representation of the results from these studiesare shown in FIG. 3.

[0280] The wild-type Cry1Ac is rapidly processed to the activeδ-endotoxin fragment that is stable for the duration of the study. Thehybrid δ-endotoxins from EG11063 and EG11074 are also processed toactive δ-endotoxin fragments which are stable for the duration of thestudy. The processing of the EG11063 δ-endotoxin occurs at a slower rateand a higher percentage of this active δ-endotoxin fragment remains ateach time point. Although the hybrid δ-endotoxins from EG11060 andEG11062 are process to active δ-endotoxin fragments, these fragments aremore susceptible to further cleavage and degrade at various rates duringthe course of the study. The 5′ exchange points between cry1Ac and cry1Ffor the EG11062 and EG11063 δ-endotoxins result in toxins that differ byonly 21 amino acid residues (see FIG. 1). However, the importance ofmaintaining Cry1Ac sequences at these positions is evident by the morerapid degradation of the EG11062 δ-endotoxin. These data demonstratethat different hybrid δ-endotoxins constructed using the same parentalδ-endotoxins can vary significantly in biochemical characteristics suchas proteotytic stability.

6.4 Example 4 Bioactivity of the Hybrid δ-Endotoxins

[0281]B. thuringiensis cultures expressing the desired δ-endotoxin weregrown until fully sporulated and lysed and washed as described inExample 2. The δ-endotoxin levels for each culture were quantified bySDS-PAGE as described (Baum et al., 1990). In the case of bioassayscreens, a single appropriate concentration of each washed δ-endotoxinculture was topically applied to 32 wells containing 1.0 ml artificialdiet per well (surface area of 175 mm²). A single neonate larvae wasplaced in each of the treated wells and the tray covered by a clearperforated mylar sheet. Larvae mortality was scored after 7 days offeeding and percent mortality expressed as the ratio of the number ofdead larvae to the total number of larvae treated, 32.

[0282] In the case of LC₅₀ determinations (δ-endotoxin concentrationgiving 50% mortality), δ-endotoxins were purified from the B.thuringiensis cultures and quantified as described by Chambers et al.(1991). Eight concentrations of the δ-endotoxins were prepared by serialdilution in 0.005% Triton X-100® and each concentration was topicallyapplied to wells containing 1.0 ml of artificial diet. Larvae mortalitywas scored after 7 days of feeding (32 larvae for each δ-endotoxinconcentration). In all cases the diluent served as the control.

[0283] A comparison of the Cry1A/Cry1F hybrid toxins by bioassay screensis shown in Table 4. The hybrid δ-endotoxins from strains EG11063 andEG11074 maintain the activities of the parental Cry1Ac and Cry1Fδ-endotoxins. Furthermore, the hybrid δ-endotoxin from EG11735 maintainsthe activity of its parental Cry1Ab and Cry1F δ-endotoxins. Theδ-endotoxins produce by strains EG11061, EG11062, EG11071, and EG11073have no insecticidal activity on the insect larvae tested despite 1)being comprised of at least one parental δ-endotoxin that is activeagainst the indicated larvae and 2) forming stable, well-definedcrystals in B. thuringiensis. These results demonstrate theunpredictable nature of hybrid toxin constructions.

[0284] For the data in Table 4. All strains were tested as washedsporulated cultures. For each insect tested, equivalent amounts ofδ-endotoxins were used and insecticidal activity was based on the strainshowing the highest percent mortality (++++). TABLE 4 BIOASSAY SCREENSOF HYBRID CRY1A/CRY1F δ-ENDOTOXINS S. Strain frugiperda S. exigua H.virescens H. zea O. nubilalis Cry1Ac − − ++++ ++++ +++ Cry1F ++++ ++ ++++ ++ Cry1Ab ++ + +++ ++ +++ EG11060 − − − − − EG11062 − − − − − EG11063++++ ++++ +++ +++ ++++ EG11071 − − − − − EG11073 − − − − − EG11074 ++++++++ +++ +++ ++++ EG11090 − +++ − − − EG11091 ++++ ++++ − − N.D. EG11092++++ ++++ +++ +++ N.D. EG11735 ++++ ++++ +++ +++ N.D. EG11751 N.D.^(a)++++ N.D. ++++ N.D.

[0285] The δ-endotoxins described in FIG. 1 and that demonstratedinsecticidal activity in bioassay screens were tested as purifiedcrystals to determine their LC₅₀ (see Table 5). The δ-endotoxinspurified from strains EG11063, EG11074, EG11091, and EG11735 all showincreased armyworm (S. frugiperda and S. exigua) activity compared toany of the wild-type δ-endotoxins tested. The EG11063 and EG11074δ-endotoxins would yield identical active toxin fragments (FIG. 1B)which is evident by their similar LC50 values on the insects examined.An unexpected result evident from these data is that a hybridδ-endotoxin such as EG11063, EG11092, EG11074, EG11735, or EG11751 canretain the activity of their respective parental δ-endotoxins, and,against certain insects such as S. exigua, can have activity far betterthan either parental δ-endotoxin. This broad range of insecticidalactivity at doses close to or lower than the parental δ-endotoxins,along with the wild-type level of toxin production (Example 2), makethese proteins particularly suitable for production in B. thuringiensis.Although the EG11091 derived δ-endotoxin has better activity against S.frugiperda and S. exigua than its parental δ-endotoxins, it has lost theH. virescens and H. zea activity attributable to its Cry1Ac parent. Thisrestricted host range along with lower toxin yield observed for theEG11091 δ-endotoxin (Example 2) make it less amenable to production inB. thuringiensis. TABLE 5 LC₅₀ VALUES FOR THE PURIFIED HYBRIDδ-ENDOTOXIN^(A) S. H. Toxin frugiperda S. exigua virescens H. zea O.nubilalis Cry1Ac >10000 >10000 9 100 23 Cry1Ab 1435 4740 118 400 17Cry1C >10000 490 >10000 >10000 >10000 Cry1F 1027 3233 54 800 51 EG11063550 114 33 80 7 (Cry1Ac/1F) EG11074 468 77 25 76 9 (Cry1Ac/1F) EG1109121 21 219 >10000 N.D.^(a) (Cry1Ac/1C)

[0286] In Table 5, the LC₅₀ values are expressed in nanograms ofpurified δ-endotoxin per well (175 mm²) and are the composite values for2 to 6 replications. nd=not determined. TABLE 6 DNA EXCHANGE SITES FORCRY1 HYBRID δ-ENDOTOXINS Plasmid SEQ ID NO: 5′ Exchange Site SEQ ID NO:3′ Exchange Site pEG1065 15 TATCCAATTCGAACGTCATC 21 ACTACCAGGTACCTTTGATGPEG1067 16 TTTAGTCATCGATTAAATCA 21 ACTACCAGGTACCTTTGATG PEG1068 17ATAATAAGAGCTCCAATGTT 21 ACTACCAGGTACCTTTGATG PEG1070 15TATCCAATTCGAACGTCATC 19 TCATGGAGAGCTCCTATGTT PEG1072 16TTTAGTCATCGATTAAATCA 19 TCATGGAGAGCTCCTATGTT PEG1074 15TATCCAATTCGAACGTCATC 35 TGCAACACTCGAGGCTGAAT PEG1076 16TTTAGTCATCGATTAAATCA 35 TGCAACACTCGAGGCTGAAT PEG1077 17ATAATAAGAGCTCCAATGTT 35 TGCAACACTCGAGGCTGAAT PEG1088 18TACATCGTAGTGCAACTCTT 22 ACTACCGGGTACCTTTGATA PEG1089 19TCATGGAGAGCTCCTATGTT — NA PEG1091 20 TTAACAAGAGCTCCTATGTT — NA PEG109218 TACATCGTAGTGCAACTCTT — NA PEG1093 — ND^(b) 21 ACTACCAGGTACCTTTGATGPEG365 17 ATAATAAGAGCTCCAATGTT 21 ACTACCAGGTACCTTTGATG PEG378 32TCAAATACCATTGGTAAAAG 21 ACTACCAGGTACCTTTGATG PEG381 32TCAAATACCATTGGTAAAAG 35 TGCAACACTCGAGGCTGAAT

[0287] Table 6 describes the DNA surrounding the 5′ and 3′ exchangepoints for the hybrid δ-endotoxins which are pertinent to the presentinvention. As evident by the SEQ ID NO, certain hybrid δ-endotoxinsshare exchange sites.

[0288] To examine the effect of other small changes in the exchange sitechosen for hybrid endotoxin construction, the activity of EG11751 andEG11063 on S. exigua and H. zea were compared (Table 7). The dataclearly show that hybrid δ-endotoxin improvements can be made byaltering the exchange site between the two parental δ-endotoxins. Inthis example, the exchange site in the EG11751 δ-endotoxin was moved 75base pairs 3′ compared to the EG11063 δ-endotoxin and results inimproved insecticidal activity. Although no significant improvement inS. exigua activity is observed between EG11063 and EG11751, asignificant improvement in H. zea activity of almost 4-fold is observedfor EG11751. It is important to note that improvements in hybridδ-endotoxin bioactivity by altering exchange sites is unpredictable. Inthe case of EG11062, moving the exchange site 63 base pairs 5′ of theEG11063 exchange site abolishes insecticidal activity as shown in Table7. TABLE 7 BIOACTIVITY OF EG11063 AND EG11751 LC₅₀ Values for WashedSporulated Cultures B. thuringiensis Strain S. exigua H. zea EG11063 10638 EG11751 90 10

[0289] To further examine the effect of changes in the exchange site forhybrid δ-endotoxins, the hybrid δ-endotoxin encoded by pEG381 wascompared to those encoded by pEG378 and pEG1068. In this example, the 3′exchange site for the pEG381 encoded hybrid δ-endotoxin was moved 340base pairs 5′ compared to the pEG378 hybrid δ-endotoxin. The data inTable 7 show that this change results in an increase in S. frugiperdaactivity compared to the pEG378 and pEG1066 encoded δ-endotoxins whilemaintaining the increased activity that was observed for the pEG378encoded δ-endotoxin over the pEG1068 encoded δ-endotoxin (see Table 6).This result is unexpected since the activated toxin resulting from theproteolysis of the encoded δ-endotoxins from pEG378 and pEG381 should beidentical. This example further demonstrates that exchange sites withinthe protoxin fragment of δ-endotoxins can have a profound effect oninsecticidal activity. TABLE 8 BIOACTIVITY OF TOXINS ENCODED BY pEG378,pEG381 AND pEG1068 LC₅₀ Values for Purified Crystals Plasmid S.frugiperda T. ni H. zea P. xylostella pEG378 464 57.7 37.5 3.02 pEG381274 56.0 36.6 2.03 pEG1068 476 66.7 72.7 3.83

6.5.1 Example 5A Activity of the Hybrid Toxins on Additional Pests

[0290] The toxins of the present invention were also assayed againstadditional pests, including the southwestern corn borer and two pestsactive against soybean. Toxin proteins were solubilized, added to dietand bioassayed against target pests. The hybrid toxins showed veryeffective control of all three pests. TABLE 9 LC₅₀ AND EC₅₀ RANGES OFHYBRID TOXINS ON SOUTHWESTERN CORN BORER^(1,2) EG11063 EG11074 EG11091EG11751 LC₅₀ 20 10-20 10-20 10-20 EC₅₀ 0.2-2   0.2-2   0.2-2   0.2-2  

[0291] TABLE 10 LC₅₀ VALUES OF CHIMERIC CRYSTAL PROTEINS ON SOYBEANPESTS¹ Pest EG11063 EG11074 EG11091 EG11751 EG11768 Velvetbean 0.9 0.60.3 0.1 0.06 caterpillar¹ Soybean 0.9 0.8 0.6 0.7 0.2 looper

6.5.1 Example 5B Activity of the Hybrid Toxins on Additional Pests

[0292] Studies were also conducted to characterize the relativeactivities of Cry1Ac, Cry2Ab2, Cry1Fa, Cry1Ca, and selected chimerictoxins against lepidopteran cotton insects in diet bioassays usingpurified proteins. The bollworms (cotton bollworm, Helicoverpa zea; pinkbollworm, Pectinophora gossypiella; tobacco budworm, Heliothisvirescens, Helicoverpa armigera and Earias vitella) and armyworms(Spodoptera exigua, Spodoptera frugiperda, and Spodoptera litura) wereused as test insects for these studies. The activity profile of fourprimary toxins—Cry1Ac, Cry2Ab2, Cry1Fa, and Cry1Ca and those of thehybrid toxins of Cry1Ac and Cry1Fa (EG11768, EG11751, EG11074, andEG11063) were determined in laboratory bioassays.

[0293] Laboratory reared H. virescens, H. zea, S. exigua, and S.frugiperda were obtained from Ecogen, Inc., Langhorne, Pa. and P.gossypyella were obtained from the insect rearing facility at theWestern Cotton Research Laboratory, Phoenix, Ariz. All insects used inthe studies had been reared in laboratories in the absence of anyinsecticidal pressure for over 20 generations. H. armigera, E. vitella,and S. litura insects were reared at the Monsanto facility at Bangalore,India.

[0294] Recombinant strains of Bacillus thuringiensis were used toexpress the primary toxins, Cry1Ac, Cry2Ab2, Cry1Ca, Cry1Fa, and fivehybrid toxins, EG11768, EG11751, EG11074, and EG11063. The toxins werethen isolated and purified from sporulated lysed cultures utilizingstandard procedures (Donovan et. al. 1992, Malvar et. al., 1994). Thecrystalline preparations of the proteins were then treated with high pHbuffer to solubilize the proteins after which they were run on SDS PAGEgels (4-20% acrylamide) and quantified against bovine serum albumin(BSA) standard (Dankocsik et. al. 1990).

[0295] Dose-response studies on the susceptibility of the differentinsect species to various toxins were performed by diet incorporation(Stone et al. 1989). A series of 6 to 8 concentrations prepared byserial dilution was used in each instance. Neonates were infested ontothe diet. Mortality and weight measurements were recorded seven daysafter infestation. Larvae that were dead or were still at the neonatestage were considered dead in tabulating larval responses to theindividual proteins. Concentration-mortality regressions were estimatedassuming the probit software model (JMP Statistical Discovery Software1995, SAS Institute, Cary, N.C.). Results were expressed as LC₅₀s inμg/ml diet.

[0296] The results obtained from several replicated experiments aresummarized in Tables 11-14. Based on LC₅₀ values, the primary toxinsexhibit insecticidal activity that differ from the hybrid toxins. Forexample, Cry1Ac has excellent to good activity on all bollworm speciesand little or no activity on armyworms; and Cry1Fa, is not toxic to H.zea (CBW), but has good activity on other more susceptible bollwormspecies (TBW and PBW) and armyworms (BAW and FAW).

[0297] In contrast, the Cry1Ac/1Fa hybrid toxins (EG11768, EG11751,EG11074, EG11063) have excellent to good activity on all of the testedLepidopteran pests. Thus, the hybrid toxins have insecticidal activityover a broader host range than either of the individual parent proteinsfrom which they were engineered. TABLE 11 ACTIVITY OF PRIMARY AND HYBRIDTOXINS ON LEPIDOPTERAN INSECTS (U.S.) TBW PBW CBW BAW FAW Primary ToxinsCry1Ac ++++ ++++ +++ −−− −−− Cry2Ab2 ++++ ++++ ++ + (+) Cry1Fa +++ +++−−− +++ +++ Cry1Ca −−− −−− +++ −−− Hybrid Toxins Cry1Ac/F ++++ ++++ ++++++ +++ (EG11768) Cry1Ac/F ++++ ++++ +++ +++ +++ (EG11751) Cry1Ac/F ++++++++ +++ +++ +++ (EG11074) Cry1Ac/F ++++ ++++ +++ +++ +++ (EG11063)

[0298] TABLE 12 ACTIVITY OF PRIMARY AND HYBRID TOXINS ON LEPIDOPTERANINSECTS (INDIA) CBW SBW CLW Primary Toxins Cry1Ac +++ ++++ + Cry2Ab2 +++Hybrid Toxins Cry1Ac/F +++ ++++ +++ (EG11768) Cry1Ac/F +++ ++++ +++(EG11751)

[0299] TABLE 13 ACTIVITY OF PRIMARY AND HYBRID TOXINS ON COTTON PESTS(U.S.) TBW CBW PBW BAW FAW Primary Toxins Cry1Ac 0.02 2.110.01 >>100 >>100 Cry2Ab2 0.44 16.75 0.04 43.81 76.31 Cry1Fa 0.61 >>1002.24 4.73 3.81 Cry1Ca >>20 >>100 5.49 >>100 Hybrid Toxins Cry1Ac/F 0.042.26 0.01 1.93 3.99 (EG11768) Cry1Ac/F 0.16 4.36 0.03 2.87 2.78(EG11751) Cry1Ac/F 0.2 9.14 0.02 2.15 0.87 (EG11074) Cry1Ac/F 0.23 8.650.05 3.42 1.033 (EG11063)

[0300] TABLE 14 ACTIVITY OF PRIMARY AND HYBRID TOXINS ON COTTON PESTS(INDIA) Cry1Ac Cry2Ab EG11768 EG11751 EG11074 EG11063 CBW 0.466 0.9951.79 2.324 2.86 SBW 0.263 2.976 0.265 0.044 0.259 0.142 CLW 40 0.9 2.4

6.6 Example 6 Amino Acid Sequences of the Novel Crystal Proteins 6.6.1Amine Acid Sequence of the EG11063 Crystal Protein (SEQ ID NO:10)

[0301]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.6.2 Amino Acid Sequence of the EG11074 Crystal Protein (SEQ ID NO:12)

[0302]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsnValThrAspTyrHisIleAspGlnValSerAsnLeuValThrTyrLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSerAsnPheLysAspIleAsnArgGlnProGluArgGlyTrpGlyGlySerThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.6.3 Amino Acid Sequence of the EG11735 Crystal Protein (SEQ ID NO:14)

[0303]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluGlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.6.4 Amino Acid Sequence of the EG11092 Crystal Protein (SEQ ID NO:26)

[0304]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsrlArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.6.5 Amino Acid Sequence of the EG11751 Crystal Protein (SEQ ID NO:28)

[0305]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsnIleIleAlaSerAspSerIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.6.6 Amino Acid Sequence of the EG11091 Crystal Protein (SEQ ID NO:30)

[0306]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGlnGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluNetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlrArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaThrLeuThrAsnThrIleAspProGluArgIleAsnGlnIleProLeuValLysGlyPheArgValTrpGlyGlyThrSerValIleThrGlyProGlyPheThrGlyGlyAspIleLeuArgArgAsnThrPheGlyAspPheValSerLeuGlnValAsnIleAsnSerProIleThrGlnArgTyrArgLeuArgPheArgTyrAlaSerSerArgAspAlaArgValIleValLeuThrGlyAlaAlaSerThrGlyValGlyGlyGlnValSerValAsnMetProLeuGlnLysThrMetGluIleGlyGluAsnLeuThrSerArgThrPheArgTyrThrAspPheSerAsnProPheSerPheArgAlaAsnProAspIleIleGlyIleSerGluGlnProLeuPheGlyAlaGlySerIleSerSerGlyGluLeuTyrIleAspLysIleGluIleIleLeuAlaAspAlaThrPheGluAlaGluSerAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheArgGlyIleAsnArgGlnProAspArgGlyTrpArgGlySerThrAspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrValAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGluLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluIleValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheThrLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuLeuGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGlnLeuGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspArgLeuGlnValAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisArgIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaTyrSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuLeuCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeuValIleProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAspAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThrValThrCysAsnAsnTyrThrGlyThrGlnGluGluTyrGluGlyThrTyrThrSerArgAsnGlnGlyTyrAspGluAlaTyrGlyAsnAsnProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluSerAsnArgGlyTyrGlyAspTyrThrProLeuProAlaGlyTyrValThrLysAspLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.6.7 Amino Acid Sequence of the EG1768 Crystal Protein (SEQ ID NO:34)

[0307]MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsnIleIleAlaSerAspSerIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsnValThrAspTyrHisIleAspGlnValSerAsnLeuValThrTyrLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSerAsnPheLysAspIleAsnArgGlnProGluArgGlyTrpGlyGlySerThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePIleGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu

6.7 Example 7 DNA Sequences Encoding the Novel Crystal Proteins 6.7.1DNA Sequence Encoding the EG11063 Crystal Protein (SEQ ID NO:9)

[0308] ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531

6.7.2 DNA Sequence Encoding the EG11074 Crystal Protein (SEQ ID NO:11)

[0309] ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA CTC GAG GCT GAA TAT AAT CTG GAA AGA GCG CAG AAG GCG GTG1872 AAT GCG CTG TTT ACG TCT ACA AAC CAA CTA GGG CTA AAA ACA AAT GTA1920 ACG GAT TAT CAT ATT GAT CAA GTG TCC AAT TTA GTT ACG TAT TTA TCG1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT2064 TTC AAA GAC ATT AAT AGG CAA CCA GAA CGT GGG TGG GGC GGA AGT ACA2112 GGG ATT ACC ATC CAA GGA GGG GAT GAC GTA TTT AAA GAA AAT TAC GTC2160 ACA CTA TCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531

6.7.3 DNA Sequence Encoding the EG11735 Crystal Protein (SEQ ID NO:13)

[0310] ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 GGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGA GGA GAA TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531

6.7.4 DNA Sequence Encoding the EG11092 Crystal Protein (SEQ ID NO:25)

[0311] ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GPA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534

6.7.5 DNA Sequence Encoding the EG11751 Crystal Protein (SEQ ID NO:27)

[0312] ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAA TTT AAT AAT1392 ATA ATT GCA TCG GAT AGT ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534

6.7.6 DNA Sequence Encoding the EG11091 Crystal Protein (SEQ ID NO:29)

[0313] ATG GAT AAC ATT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCA ACT CTT ACA AAT1392 ACA ATT GAT CCA GAG AGA ATT AAT CAA ATA CCT TTA GTG AAA GGA TTT1440 AGA GTT TGG GGG GGC ACC TCT GTC ATT ACA GGA CCA GGA TTT ACA GGA1488 GGG GAT ATC CTT CGA AGA AAT ACC TTT GGT GAT TTT GTA TCT CTA CAA1536 GTC AAT ATT ATT TCA CCA ATT ACC CAA AGA TAC CGT TTA AGA TTT CGT1584 TAC GCT TCC AGT AGG GAT GCA CGA GTT ATA GTA TTA ACA GGA GCG GCA1632 TCC ACA GGA GTG GGA GGC CAA GTT AGT GTA AAT ATG CCT CTT CAG AAA1680 ACT ATG GAA ATA GGG GAG AAC TTA ACA TCT AGA ACA TTT AGA TAT ACC1728 GAT TTT AGT AAT CCT TTT TCA TTT AGA GCT AAT CCA GAT ATA ATT GGG1776 ATA AGT GAA CAA CCT CTA TTT GGT GCA GGT TCT ATT AGT AGC GGT GAA1824 CTT TAT ATA GAT AAA ATT GAA ATT ATT CTA GCA GAT GCA ACA TTT GAA1872 GCA GAA TCT GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCC CTG TTT1920 ACT TCT TCC AAT CAA ATC GGG TTA AAA ACC GAT GTG ACG GAT TAT CAT1968 ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA TTT TGT2016 CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA2064 CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC TTC AGA GGG ATC2112 AAT AGA CAA CCA GAC CGT GGC TGG AGA GGA AGT ACA GAT ATT ACC ATC2160 CAA GGA GGA GAT GAC GTA TTC AAA GAG AAT TAC GTC ACA CTA CCG GGT2208 ACC GTT GAT GAG TGC TAT CCA ACG TAT TTA TAT CAG AAA ATA GAT GAG2256 TCG AAA TTA AAA GCT TAT ACC CGT TAT GAA TTA AGA GGG TAT ATC GAA2304 GAT AGT CAA GAC TTA GAA ATC TAT TTG ATC CGT TAC AAT GCA AAA CAC2352 GAA ATA GTA AAT GTG CCA GGC ACG GGT TCC TTA TGG CCG CTT TCA GCC2400 CAA AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC2448 CTT GAA TGG AAT CCT GAT CTA GAT TGT TCC TGC AGA GAC GGG GAA AAA2496 TGT GCA CAT CAT TCC CAT CAT TTC ACC TTG GAT ATT GAT GTT GGA TGT2544 ACA GAC TTA AAT GAG GAC TTA GGT GTA TGG GTG ATA TTC AAG ATT AAG2592 ACG CAA GAT GGC CAT GCA AGA CTA GGG AAT CTA GAG TTT CTC GAA GAG2640 AAA CCA TTA TTA GGG GAA GCA CTA GCT CGT GTG AAA AGA GCG GAG AAG2688 AAG TGG AGA GAC AAA CGA GAG AAA CTG CAG TTG GAA ACA AAT ATT GTT2736 TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA2784 TAT GAT AGA TTA CAA GTG GAT ACG AAC ATC GCA ATG ATT CAT GCG GCA2832 GAT AAA CGC GTT CAT AGA ATC CGG GAA GCG TAT CTG CCA GAG TTG TCT2880 GTG ATT CCA GGT GTC AAT GCG GCC ATT TTC GAA GAA TTA GAG GGA CGT2928 ATT TTT ACA GCG TAT TCC TTA TAT GAT GCG AGA AAT GTC ATT AAA AAT2976 GGC GAT TTC AAT AAT GGC TTA TTA TGC TGG AAC GTG AAA GGT CAT GTA3024 GAT GTA GAA GAG CAA AAC AAC CAC CGT TCG GTC CTT GTT ATC CCA GAA3072 TGG GAG GCA GAA GTG TCA CAA GAG GTT CGT GTC TGT CCA GGT CGT GGC3120 TAT ATC CTT CGT GTC ACA GCA TAT AAA GAG GGA TAT GGA GAG GGC TGC3168 GTA ACG ATC CAT GAG ATC GAA GAC AAT ACA GAC GAA CTG AAA TTC AGC3216 AAC TGT GTA GAA GAG GAA GTA TAT CCA AAC AAC ACA GTA ACG TGT AAT3264 AAT TAT ACT GGG ACT CAA GAA GAA TAT GAG GGT ACG TAC ACT TCT CGT3312 AAT CAA GGA TAT GAC GAA GCC TAT GGT AAT AAC CCT TCC GTA CCA GCT3360 GAT TAC GCT TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3408 GAG AAT CCT TGT GAA TCT AAC AGA GGC TAT GGG GAT TAC ACA CCA CTA3456 CCG GCT GGT TAT GTA ACA AAG GAT TTA GAG TAC TTC CCA GAG ACC GAT3504 AAG GTA TGG ATT GAG ATC GGA GAA ACA GAA GGA ACA TTC ATC GTG GAT3552 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3579

6.7.7 DNA Sequence Encoding the EG11768 Crystal Protein (SEQ ID NO:33)

[0314] ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA  48 AGT AAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT  96 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAA TTT AAT AAT1392 ATA ATT GCA TCG GAT AGT ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA CTC GAG GCT GAA TAT AAT CTG GAA AGA GCG CAG AAG GCG GTG1872 AAT GCG CTG TTT ACG TCT ACA AAC CAA CTA GGG CTA AAA ACA AAT GTA1920 ACG GAT TAT CAT ATT GAT CAA GTG TCC AAT TTA GTT ACG TAT TTA TCG1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT2064 TTC AAA GAC ATT AAT AGG CAA CCA GAA CGT GGG TGG GGC GGA AGT ACA2112 GGG ATT ACC ATC CAA GGA GGG GAT GAC GTA TTT AAA GAA AAT TAC GTC2160 ACA CTA TCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534

[0315] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

7. References

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1 35 1 23 DNA Artificial sequence Synthetic Oligonucleotide 1 ggatagcactcatcaaaggt acc 23 2 27 DNA Artificial sequence Synthetic Oligonucleotide2 gaagatatcc aattcgaaca gtttccc 27 3 28 DNA Artificial sequenceSynthetic Oligonucleotide 3 catattctgc ctcgagtgtt gcagtaac 28 4 17 DNAArtificial sequence Synthetic Oligonucleotide 4 cccgatcggc cgcatgc 17 517 DNA Artificial sequence Synthetic Oligonucleotide 5 cattggagctctccatg 17 6 16 DNA Artificial sequence Synthetic Oligonucleotide 6gcactacgat gtatcc 16 7 20 DNA Artificial sequence SyntheticOligonucleotide 7 catcgtagtg caactcttac 20 8 39 DNA Artificial sequenceSynthetic Oligonucleotide 8 ccaagaaaat actagagctc ttgttaaaaa aggtgttcc39 9 3531 DNA Artificial sequence Hybrid Delta-Endotoxin 9 atg gat aacaat ccg aac atc aat gaa tgc att cct tat aat tgt tta 48 Met Asp Asn AsnPro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agt aac cctgaa gta gaa gta tta ggt gga gaa aga ata gaa act ggt 96 Ser Asn Pro GluVal Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 tac acc cca atcgat att tcc ttg tcg cta acg caa ttt ctt ttg agt 144 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 gaa ttt gtt ccc ggtgct gga ttt gtg tta gga cta gtt gat ata ata 192 Glu Phe Val Pro Gly AlaGly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 tgg gga att ttt ggt ccctct caa tgg gac gca ttt ctt gta caa att 240 Trp Gly Ile Phe Gly Pro SerGln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gaa cag tta att aac caaaga ata gaa gaa ttc gct agg aac caa gcc 288 Glu Gln Leu Ile Asn Gln ArgIle Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 att tct aga tta gaa gga ctaagc aat ctt tat caa att tac gca gaa 336 Ile Ser Arg Leu Glu Gly Leu SerAsn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 tct ttt aga gag tgg gaa gcagat cct act aat cca gca tta aga gaa 384 Ser Phe Arg Glu Trp Glu Ala AspPro Thr Asn Pro Ala Leu Arg Glu 115 120 125 gag atg cgt att caa ttc aatgac atg aac agt gcc ctt aca acc gct 432 Glu Met Arg Ile Gln Phe Asn AspMet Asn Ser Ala Leu Thr Thr Ala 130 135 140 att cct ctt ttt gca gtt caaaat tat caa gtt cct ctt tta tca gta 480 Ile Pro Leu Phe Ala Val Gln AsnTyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 tat gtt caa gct gca aattta cat tta tca gtt ttg aga gat gtt tca 528 Tyr Val Gln Ala Ala Asn LeuHis Leu Ser Val Leu Arg Asp Val Ser 165 170 175 gtg ttt gga caa agg tgggga ttt gat gcc gcg act atc aat agt cgt 576 Val Phe Gly Gln Arg Trp GlyPhe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 tat aat gat tta act aggctt att ggc aac tat aca gat tat gct gta 624 Tyr Asn Asp Leu Thr Arg LeuIle Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 cgc tgg tac aat acg ggatta gaa cgt gta tgg gga ccg gat tct aga 672 Arg Trp Tyr Asn Thr Gly LeuGlu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 gat tgg gta agg tat aatcaa ttt aga aga gaa tta aca cta act gta 720 Asp Trp Val Arg Tyr Asn GlnPhe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 tta gat atc gtt gctctg ttc ccg aat tat gat agt aga aga tat cca 768 Leu Asp Ile Val Ala LeuPhe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 att cga aca gtt tcccaa tta aca aga gaa att tat aca aac cca gta 816 Ile Arg Thr Val Ser GlnLeu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 tta gaa aat ttt gatggt agt ttt cga ggc tcg gct cag ggc ata gaa 864 Leu Glu Asn Phe Asp GlySer Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 aga agt att agg agtcca cat ttg atg gat ata ctt aac agt ata acc 912 Arg Ser Ile Arg Ser ProHis Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 atc tat acg gat gctcat agg ggt tat tat tat tgg tca ggg cat caa 960 Ile Tyr Thr Asp Ala HisArg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ata atg gct tctcct gta ggg ttt tcg ggg cca gaa ttc act ttt ccg 1008 Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 cta tat gga actatg gga aat gca gct cca caa caa cgt att gtt gct 1056 Leu Tyr Gly Thr MetGly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 caa cta ggt cagggc gtg tat aga aca tta tcg tcc act tta tat aga 1104 Gln Leu Gly Gln GlyVal Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 aga cct ttt aatata ggg ata aat aat caa caa cta tct gtt ctt gac 1152 Arg Pro Phe Asn IleGly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 ggg aca gaa tttgct tat gga acc tcc tca aat ttg cca tcc gct gta 1200 Gly Thr Glu Phe AlaTyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 tac aga aaaagc gga acg gta gat tcg ctg gat gaa ata ccg cca cag 1248 Tyr Arg Lys SerGly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 aat aac aacgtg cca cct agg caa gga ttt agt cat cga tta agc cat 1296 Asn Asn Asn ValPro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 gtt tca atgttt cgt tca ggc ttt agt aat agt agt gta agt ata ata 1344 Val Ser Met PheArg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 aga gct ccaatg ttt tct tgg acg cac cgt agt gca acc cct aca aat 1392 Arg Ala Pro MetPhe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn 450 455 460 aca att gatccg gag agg att act caa ata cca ttg gta aaa gca cat 1440 Thr Ile Asp ProGlu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His 465 470 475 480 aca cttcag tca ggt act act gtt gta aga ggg ccc ggg ttt acg gga 1488 Thr Leu GlnSer Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 gga gatatt ctt cga cga aca agt gga gga cca ttt gct tat act att 1536 Gly Asp IleLeu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 gtt aatata aat ggg caa tta ccc caa agg tat cgt gca aga ata cgc 1584 Val Asn IleAsn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 tat gcctct act aca aat cta aga att tac gta acg gtt gca ggt gaa 1632 Tyr Ala SerThr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 cgg attttt gct ggt caa ttt aac aaa aca atg gat acc ggt gac cca 1680 Arg Ile PheAla Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 ttaaca ttc caa tct ttt agt tac gca act att aat aca gct ttt aca 1728 Leu ThrPhe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 ttccca atg agc cag agt agt ttc aca gta ggt gct gat act ttt agt 1776 Phe ProMet Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 tcaggg aat gaa gtt tat ata gac aga ttt gaa ttg att cca gtt act 1824 Ser GlyAsn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 gcaaca ttt gaa gca gaa tat gat tta gaa aga gca caa aag gcg gtg 1872 Ala ThrPhe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 aatgcg ctg ttt act tct ata aac caa ata ggg ata aaa aca gat gtg 1920 Asn AlaLeu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635 640acg gat tat cat att gat caa gta tcc aat tta gtg gat tgt tta tca 1968 ThrAsp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645 650 655gat gaa ttt tgt ctg gat gaa aag cga gaa ttg tcc gag aaa gtc aaa 2016 AspGlu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670cat gcg aag cga ctc agt gat gag cgg aat tta ctt caa gat cca aac 2064 HisAla Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685ttc aaa ggc atc aat agg caa cta gac cgt ggt tgg aga gga agt acg 2112 PheLys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700gat att acc atc caa aga gga gat gac gta ttc aaa gaa aat tat gtc 2160 AspIle Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715720 aca cta cca ggt acc ttt gat gag tgc tat cca aca tat ttg tat caa 2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730735 aaa atc gat gaa tca aaa tta aaa gcc ttt acc cgt tat caa tta aga 2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745750 ggg tat atc gaa gat agt caa gac tta gaa atc tat tta att cgc tac 2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760765 aat gca aaa cat gaa aca gta aat gtg cca ggt acg ggt tcc tta tgg 2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775780 ccg ctt tca gcc caa agt cca atc gga aag tgt gga gag ccg aat cga 2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790795 800 tgc gcg cca cac ctt gaa tgg aat cct gac tta gat tgt tcg tgt agg2448 Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805810 815 gat gga gaa aag tgt gcc cat cat tcg cat cat ttc tcc tta gac att2496 Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820825 830 gat gta gga tgt aca gac tta aat gag gac cta ggt gta tgg gtg atc2544 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835840 845 ttt aag att aag acg caa gat ggg cac gca aga cta ggg aat cta gag2592 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850855 860 ttt ctc gaa gag aaa cca tta gta gga gaa gcg cta gct cgt gtg aaa2640 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865870 875 880 aga gcg gag aaa aaa tgg aga gac aaa cgt gaa aaa ttg gaa tgggaa 2688 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu885 890 895 aca aat atc gtt tat aaa gag gca aaa gaa tct gta gat gct ttattt 2736 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe900 905 910 gta aac tct caa tat gat caa tta caa gcg gat acg aat att gccatg 2784 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met915 920 925 att cat gcg gca gat aaa cgt gtt cat agc att cga gaa gct tatctg 2832 Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu930 935 940 cct gag ctg tct gtg att ccg ggt gtc aat gcg gct att ttt gaagaa 2880 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu945 950 955 960 tta gaa ggg cgt att ttc act gca ttc tcc cta tat gat gcgaga aat 2928 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala ArgAsn 965 970 975 gtc att aaa aat ggt gat ttt aat aat ggc tta tcc tgc tggaac gtg 2976 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp AsnVal 980 985 990 aaa ggg cat gta gat gta gaa gaa caa aac aac caa cgt tcggtc ctt 3024 Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser ValLeu 995 1000 1005 gtt gtt ccg gaa tgg gaa gca gaa gtg tca caa gaa gttcgt gtc 3069 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val1010 1015 1020 tgt ccg ggt cgt ggc tat atc ctt cgt gtc aca gcg tac aaggag 3114 Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu1025 1030 1035 gga tat gga gaa ggt tgc gta acc att cat gag atc gag aacaat 3159 Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn1040 1045 1050 aca gac gaa ctg aag ttt agc aac tgc gta gaa gag gaa atctat 3204 Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr1055 1060 1065 cca aat aac acg gta acg tgt aat gat tat act gta aat caagaa 3249 Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu1070 1075 1080 gaa tac gga ggt gcg tac act tct cgt aat cga gga tat aacgaa 3294 Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu1085 1090 1095 gct cct tcc gta cca gct gat tat gcg tca gtc tat gaa gaaaaa 3339 Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys1100 1105 1110 tcg tat aca gat gga cga aga gag aat cct tgt gaa ttt aacaga 3384 Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg1115 1120 1125 ggg tat agg gat tac acg cca cta cca gtt ggt tat gtg acaaaa 3429 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys1130 1135 1140 gaa tta gaa tac ttc cca gaa acc gat aag gta tgg att gagatt 3474 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile1145 1150 1155 gga gaa acg gaa gga aca ttt atc gtg gac agc gtg gaa ttactc 3519 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu1160 1165 1170 ctt atg gag gaa 3531 Leu Met Glu Glu 1175 10 1177 PRTArtificial sequence Hybrid Delta-Endotoxin 10 Met Asp Asn Asn Pro AsnIle Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu ValGlu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro GlyAla Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe GlyPro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu IleAsn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg LeuGlu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe ArgGlu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu MetArg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 IlePro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr AlaVal 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro AspSer Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr AspSer Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg GluIle Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe ArgGly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser Pro His LeuMet Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Ala His ArgGly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly ThrMet Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 Gln Leu GlyGln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg ProPhe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 GlyThr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser IleIle 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr ProThr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu ValLys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr Thr Val Val Arg GlyPro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Gly GlyPro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn Gly Gln Leu Pro GlnArg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu ArgIle Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile Phe Ala Gly Gln PheAsn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 Leu Thr Phe Gln SerPhe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 Phe Pro Met SerGln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 Ser Gly AsnGlu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 Ala ThrPhe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AsnAla Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp ProAsn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg GlySer Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys GluAsn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr ProThr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser Lys Leu Lys Ala PheThr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu Asp Ser Gln Asp LeuGlu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys His Glu Thr Val AsnVal Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu Ser Ala Gln Ser ProIle Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 Cys Ala Pro His LeuGlu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 Asp Gly Glu LysCys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 Asp Val GlyCys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 Phe LysIle Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 PheLeu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile AlaMet 915 920 925 Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu AlaTyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala IlePhe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser LeuTyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly Asp Phe Asn Asn GlyLeu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val Asp Val Glu Glu GlnAsn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val Pro Glu Trp Glu AlaGlu Val Ser Gln Glu Val Arg Val 1010 1015 1020 Cys Pro Gly Arg Gly TyrIle Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 Gly Tyr Gly Glu GlyCys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 Thr Asp Glu LeuLys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 Pro Asn AsnThr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080 Glu TyrGly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu 1085 1090 1095 AlaPro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys 1100 1105 1110Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg 1115 11201125 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys 11301135 1140 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile1145 1150 1155 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu LeuLeu 1160 1165 1170 Leu Met Glu Glu 1175 11 3531 DNA Artificial sequenceHybrid Delta-Endotoxin 11 atg gat aac aat ccg aac atc aat gaa tgc attcct tat aat tgt tta 48 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile ProTyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gta gaa gta tta ggt gga gaaaga ata gaa act ggt 96 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu ArgIle Glu Thr Gly 20 25 30 tac acc cca atc gat att tcc ttg tcg cta acg caattt ctt ttg agt 144 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln PheLeu Leu Ser 35 40 45 gaa ttt gtt ccc ggt gct gga ttt gtg tta gga cta gttgat ata ata 192 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val AspIle Ile 50 55 60 tgg gga att ttt ggt ccc tct caa tgg gac gca ttt ctt gtacaa att 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val GlnIle 65 70 75 80 gaa cag tta att aac caa aga ata gaa gaa ttc gct agg aaccaa gcc 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn GlnAla 85 90 95 att tct aga tta gaa gga cta agc aat ctt tat caa att tac gcagaa 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu100 105 110 tct ttt aga gag tgg gaa gca gat cct act aat cca gca tta agagaa 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125 gag atg cgt att caa ttc aat gac atg aac agt gcc ctt aca accgct 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala130 135 140 att cct ctt ttt gca gtt caa aat tat caa gtt cct ctt tta tcagta 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val145 150 155 160 tat gtt caa gct gca aat tta cat tta tca gtt ttg aga gatgtt tca 528 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp ValSer 165 170 175 gtg ttt gga caa agg tgg gga ttt gat gcc gcg act atc aatagt cgt 576 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn SerArg 180 185 190 tat aat gat tta act agg ctt att ggc aac tat aca gat tatgct gta 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr AlaVal 195 200 205 cgc tgg tac aat acg gga tta gaa cgt gta tgg gga ccg gattct aga 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp SerArg 210 215 220 gat tgg gta agg tat aat caa ttt aga aga gaa tta aca ctaact gta 720 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu ThrVal 225 230 235 240 tta gat atc gtt gct ctg ttc ccg aat tat gat agt agaaga tat cca 768 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg ArgTyr Pro 245 250 255 att cga aca gtt tcc caa tta aca aga gaa att tat acaaac cca gta 816 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr AsnPro Val 260 265 270 tta gaa aat ttt gat ggt agt ttt cga ggc tcg gct cagggc ata gaa 864 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln GlyIle Glu 275 280 285 aga agt att agg agt cca cat ttg atg gat ata ctt aacagt ata acc 912 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn SerIle Thr 290 295 300 atc tat acg gat gct cat agg ggt tat tat tat tgg tcaggg cat caa 960 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser GlyHis Gln 305 310 315 320 ata atg gct tct cct gta ggg ttt tcg ggg cca gaattc act ttt ccg 1008 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu PheThr Phe Pro 325 330 335 cta tat gga act atg gga aat gca gct cca caa caacgt att gtt gct 1056 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln ArgIle Val Ala 340 345 350 caa cta ggt cag ggc gtg tat aga aca tta tcg tccact tta tat aga 1104 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser ThrLeu Tyr Arg 355 360 365 aga cct ttt aat ata ggg ata aat aat caa caa ctatct gtt ctt gac 1152 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu SerVal Leu Asp 370 375 380 ggg aca gaa ttt gct tat gga acc tcc tca aat ttgcca tcc gct gta 1200 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu ProSer Ala Val 385 390 395 400 tac aga aaa agc gga acg gta gat tcg ctg gatgaa ata ccg cca cag 1248 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp GluIle Pro Pro Gln 405 410 415 aat aac aac gtg cca cct agg caa gga ttt agtcat cga tta agc cat 1296 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser HisArg Leu Ser His 420 425 430 gtt tca atg ttt cgt tca ggc ttt agt aat agtagt gta agt ata ata 1344 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser SerVal Ser Ile Ile 435 440 445 aga gct cca atg ttt tct tgg acg cac cgt agtgca acc cct aca aat 1392 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser AlaThr Pro Thr Asn 450 455 460 aca att gat ccg gag agg att act caa ata ccattg gta aaa gca cat 1440 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro LeuVal Lys Ala His 465 470 475 480 aca ctt cag tca ggt act act gtt gta agaggg ccc ggg ttt acg gga 1488 Thr Leu Gln Ser Gly Thr Thr Val Val Arg GlyPro Gly Phe Thr Gly 485 490 495 gga gat att ctt cga cga aca agt gga ggacca ttt gct tat act att 1536 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly ProPhe Ala Tyr Thr Ile 500 505 510 gtt aat ata aat ggg caa tta ccc caa aggtat cgt gca aga ata cgc 1584 Val Asn Ile Asn Gly Gln Leu Pro Gln Arg TyrArg Ala Arg Ile Arg 515 520 525 tat gcc tct act aca aat cta aga att tacgta acg gtt gca ggt gaa 1632 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr ValThr Val Ala Gly Glu 530 535 540 cgg att ttt gct ggt caa ttt aac aaa acaatg gat acc ggt gac cca 1680 Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr MetAsp Thr Gly Asp Pro 545 550 555 560 tta aca ttc caa tct ttt agt tac gcaact att aat aca gct ttt aca 1728 Leu Thr Phe Gln Ser Phe Ser Tyr Ala ThrIle Asn Thr Ala Phe Thr 565 570 575 ttc cca atg agc cag agt agt ttc acagta ggt gct gat act ttt agt 1776 Phe Pro Met Ser Gln Ser Ser Phe Thr ValGly Ala Asp Thr Phe Ser 580 585 590 tca ggg aat gaa gtt tat ata gac agattt gaa ttg att cca gtt act 1824 Ser Gly Asn Glu Val Tyr Ile Asp Arg PheGlu Leu Ile Pro Val Thr 595 600 605 gca aca ttt gaa gca gaa tat gat ttagaa aga gca caa aag gcg gtg 1872 Ala Thr Phe Glu Ala Glu Tyr Asp Leu GluArg Ala Gln Lys Ala Val 610 615 620 aat gcg ctg ttt act tct ata aac caaata ggg ata aaa aca gat gtg 1920 Asn Ala Leu Phe Thr Ser Ile Asn Gln IleGly Ile Lys Thr Asp Val 625 630 635 640 acg gat tat cat att gat caa gtatcc aat tta gtg gat tgt tta tca 1968 Thr Asp Tyr His Ile Asp Gln Val SerAsn Leu Val Asp Cys Leu Ser 645 650 655 gat gaa ttt tgt ctg gat gaa aagcga gaa ttg tcc gag aaa gtc aaa 2016 Asp Glu Phe Cys Leu Asp Glu Lys ArgGlu Leu Ser Glu Lys Val Lys 660 665 670 cat gcg aag cga ctc agt gat gagcgg aat tta ctt caa gat cca aac 2064 His Ala Lys Arg Leu Ser Asp Glu ArgAsn Leu Leu Gln Asp Pro Asn 675 680 685 ttc aaa ggc atc aat agg caa ctagac cgt ggt tgg aga gga agt acg 2112 Phe Lys Gly Ile Asn Arg Gln Leu AspArg Gly Trp Arg Gly Ser Thr 690 695 700 gat att acc atc caa aga gga gatgac gta ttc aaa gaa aat tat gtc 2160 Asp Ile Thr Ile Gln Arg Gly Asp AspVal Phe Lys Glu Asn Tyr Val 705 710 715 720 aca cta cca ggt acc ttt gatgag tgc tat cca aca tat ttg tat caa 2208 Thr Leu Pro Gly Thr Phe Asp GluCys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 aaa atc gat gaa tca aaa ttaaaa gcc ttt acc cgt tat caa tta aga 2256 Lys Ile Asp Glu Ser Lys Leu LysAla Phe Thr Arg Tyr Gln Leu Arg 740 745 750 ggg tat atc gaa gat agt caagac tta gaa atc tat tta att cgc tac 2304 Gly Tyr Ile Glu Asp Ser Gln AspLeu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 aat gca aaa cat gaa aca gtaaat gtg cca ggt acg ggt tcc tta tgg 2352 Asn Ala Lys His Glu Thr Val AsnVal Pro Gly Thr Gly Ser Leu Trp 770 775 780 ccg ctt tca gcc caa agt ccaatc gga aag tgt gga gag ccg aat cga 2400 Pro Leu Ser Ala Gln Ser Pro IleGly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 tgc gcg cca cac ctt gaatgg aat cct gac tta gat tgt tcg tgt agg 2448 Cys Ala Pro His Leu Glu TrpAsn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 gat gga gaa aag tgt gcccat cat tcg cat cat ttc tcc tta gac att 2496 Asp Gly Glu Lys Cys Ala HisHis Ser His His Phe Ser Leu Asp Ile 820 825 830 gat gta gga tgt aca gactta aat gag gac cta ggt gta tgg gtg atc 2544 Asp Val Gly Cys Thr Asp LeuAsn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 ttt aag att aag acg caagat ggg cac gca aga cta ggg aat cta gag 2592 Phe Lys Ile Lys Thr Gln AspGly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 ttt ctc gaa gag aaa ccatta gta gga gaa gcg cta gct cgt gtg aaa 2640 Phe Leu Glu Glu Lys Pro LeuVal Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880 aga gcg gag aaa aaatgg aga gac aaa cgt gaa aaa ttg gaa tgg gaa 2688 Arg Ala Glu Lys Lys TrpArg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895 aca aat atc gtt tataaa gag gca aaa gaa tct gta gat gct tta ttt 2736 Thr Asn Ile Val Tyr LysGlu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910 gta aac tct caa tatgat caa tta caa gcg gat acg aat att gcc atg 2784 Val Asn Ser Gln Tyr AspGln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925 att cat gcg gca gataaa cgt gtt cat agc att cga gaa gct tat ctg 2832 Ile His Ala Ala Asp LysArg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940 cct gag ctg tct gtgatt ccg ggt gtc aat gcg gct att ttt gaa gaa 2880 Pro Glu Leu Ser Val IlePro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 tta gaa ggg cgtatt ttc act gca ttc tcc cta tat gat gcg aga aat 2928 Leu Glu Gly Arg IlePhe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 gtc att aaa aatggt gat ttt aat aat ggc tta tcc tgc tgg aac gtg 2976 Val Ile Lys Asn GlyAsp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 aaa ggg cat gtagat gta gaa gaa caa aac aac caa cgt tcg gtc ctt 3024 Lys Gly His Val AspVal Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 gtt gtt ccggaa tgg gaa gca gaa gtg tca caa gaa gtt cgt gtc 3069 Val Val Pro Glu TrpGlu Ala Glu Val Ser Gln Glu Val Arg Val 1010 1015 1020 tgt ccg ggt cgtggc tat atc ctt cgt gtc aca gcg tac aag gag 3114 Cys Pro Gly Arg Gly TyrIle Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 gga tat gga gaa ggttgc gta acc att cat gag atc gag aac aat 3159 Gly Tyr Gly Glu Gly Cys ValThr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 aca gac gaa ctg aag tttagc aac tgc gta gaa gag gaa atc tat 3204 Thr Asp Glu Leu Lys Phe Ser AsnCys Val Glu Glu Glu Ile Tyr 1055 1060 1065 cca aat aac acg gta acg tgtaat gat tat act gta aat caa gaa 3249 Pro Asn Asn Thr Val Thr Cys Asn AspTyr Thr Val Asn Gln Glu 1070 1075 1080 gaa tac gga ggt gcg tac act tctcgt aat cga gga tat aac gaa 3294 Glu Tyr Gly Gly Ala Tyr Thr Ser Arg AsnArg Gly Tyr Asn Glu 1085 1090 1095 gct cct tcc gta cca gct gat tat gcgtca gtc tat gaa gaa aaa 3339 Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser ValTyr Glu Glu Lys 1100 1105 1110 tcg tat aca gat gga cga aga gag aat ccttgt gaa ttt aac aga 3384 Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys GluPhe Asn Arg 1115 1120 1125 ggg tat agg gat tac acg cca cta cca gtt ggttat gtg aca aaa 3429 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr ValThr Lys 1130 1135 1140 gaa tta gaa tac ttc cca gaa acc gat aag gta tggatt gag att 3474 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile GluIle 1145 1150 1155 gga gaa acg gaa gga aca ttt atc gtg gac agc gtg gaatta ctc 3519 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu1160 1165 1170 ctt atg gag gaa 3531 Leu Met Glu Glu 1175 12 1177 PRTArtificial sequence Hybrid Delta-Endotoxin 12 Met Asp Asn Asn Pro AsnIle Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu ValGlu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro GlyAla Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe GlyPro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu IleAsn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg LeuGlu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe ArgGlu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu MetArg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 IlePro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr AlaVal 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro AspSer Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr AspSer Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg GluIle Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe ArgGly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser Pro His LeuMet Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Ala His ArgGly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly ThrMet Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 Gln Leu GlyGln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg ProPhe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 GlyThr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser IleIle 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr ProThr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu ValLys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr Thr Val Val Arg GlyPro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Gly GlyPro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn Gly Gln Leu Pro GlnArg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu ArgIle Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile Phe Ala Gly Gln PheAsn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 Leu Thr Phe Gln SerPhe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 Phe Pro Met SerGln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 Ser Gly AsnGlu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 Ala ThrPhe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AsnAla Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp ProAsn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg GlySer Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys GluAsn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr ProThr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser Lys Leu Lys Ala PheThr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu Asp Ser Gln Asp LeuGlu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys His Glu Thr Val AsnVal Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu Ser Ala Gln Ser ProIle Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 Cys Ala Pro His LeuGlu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 Asp Gly Glu LysCys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 Asp Val GlyCys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 Phe LysIle Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 PheLeu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile AlaMet 915 920 925 Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu AlaTyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala IlePhe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser LeuTyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly Asp Phe Asn Asn GlyLeu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val Asp Val Glu Glu GlnAsn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val Pro Glu Trp Glu AlaGlu Val Ser Gln Glu Val Arg Val 1010 1015 1020 Cys Pro Gly Arg Gly TyrIle Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 Gly Tyr Gly Glu GlyCys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 Thr Asp Glu LeuLys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 Pro Asn AsnThr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080 Glu TyrGly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu 1085 1090 1095 AlaPro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys 1100 1105 1110Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg 1115 11201125 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys 11301135 1140 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile1145 1150 1155 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu LeuLeu 1160 1165 1170 Leu Met Glu Glu 1175 13 3531 DNA Artificial sequenceHybrid Delta-Endotoxin 13 atg gat aac aat ccg aac atc aat gaa tgc attcct tat aat tgt tta 48 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile ProTyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gta gaa gta tta ggt gga gaaaga ata gaa act ggt 96 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu ArgIle Glu Thr Gly 20 25 30 tac acc cca atc gat att tcc ttg tcg cta acg caattt ctt ttg agt 144 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln PheLeu Leu Ser 35 40 45 gaa ttt gtt ccc ggt gct gga ttt gtg tta gga cta gttgat ata ata 192 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val AspIle Ile 50 55 60 tgg gga att ttt ggt ccc tct caa tgg gac gca ttt ctt gtacaa att 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val GlnIle 65 70 75 80 gaa cag tta att aac caa aga ata gaa gaa ttc gct agg aaccaa gcc 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn GlnAla 85 90 95 att tct aga tta gaa gga cta agc aat ctt tat caa att tac gcagaa 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu100 105 110 tct ttt aga gag tgg gaa gca gat cct act aat cca gca tta agagaa 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125 gag atg cgt att caa ttc aat gac atg aac agt gcc ctt aca accgct 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala130 135 140 att cct ctt ttt gca gtt caa aat tat caa gtt cct ctt tta tcagta 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val145 150 155 160 tat gtt caa gct gca aat tta cat tta tca gtt ttg aga gatgtt tca 528 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp ValSer 165 170 175 gtg ttt gga caa agg tgg gga ttt gat gcc gcg act atc aatagt cgt 576 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn SerArg 180 185 190 tat aat gat tta act agg ctt att ggc aac tat aca gat tatgct gta 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr AlaVal 195 200 205 cgc tgg tac aat acg gga tta gaa cgt gta tgg gga ccg gattct aga 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp SerArg 210 215 220 gat tgg gta agg tat aat caa ttt aga aga gaa tta aca ctaact gta 720 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu ThrVal 225 230 235 240 tta gat atc gtt gct ctg ttc ccg aat tat gat agt agaaga tat cca 768 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg ArgTyr Pro 245 250 255 att cga aca gtt tcc caa tta aca aga gaa att tat acaaac cca gta 816 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr AsnPro Val 260 265 270 tta gaa aat ttt gat ggt agt ttt cga ggc tcg gct cagggc ata gaa 864 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln GlyIle Glu 275 280 285 aga agt att agg agt cca cat ttg atg gat ata ctt aacagt ata acc 912 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn SerIle Thr 290 295 300 atc tat acg gat gct cat agg ggt tat tat tat tgg tcaggg cat caa 960 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser GlyHis Gln 305 310 315 320 ata atg gct tct cct gta ggg ttt tcg ggg cca gaattc act ttt ccg 1008 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu PheThr Phe Pro 325 330 335 cta tat gga act atg gga aat gca gct cca caa caacgt att gtt gct 1056 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln ArgIle Val Ala 340 345 350 caa cta ggt cag ggc gtg tat aga aca tta tcg tccact tta tat aga 1104 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser ThrLeu Tyr Arg 355 360 365 aga cct ttt aat ata ggg ata aat aat caa caa ctatct gtt ctt gac 1152 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu SerVal Leu Asp 370 375 380 ggg aca gaa ttt gct tat gga acc tcc tca aat ttgcca tcc gct gta 1200 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu ProSer Ala Val 385 390 395 400 tac aga aaa agc gga acg gta gat tcg ctg gatgaa ata ccg cca cag 1248 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp GluIle Pro Pro Gln 405 410 415 aat aac aac gtg cca cct agg caa gga ttt agtcat cga tta agc cat 1296 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser HisArg Leu Ser His 420 425 430 gtt tca atg ttt cgt tca ggc ttt agt aat agtagt gta agt ata ata 1344 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser SerVal Ser Ile Ile 435 440 445 aga gct cca atg ttt tct tgg acg cac cgt agtgca acc cct aca aat 1392 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser AlaThr Pro Thr Asn 450 455 460 aca att gat ccg gag agg att act caa ata ccattg gta aaa gca cat 1440 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro LeuVal Lys Ala His 465 470 475 480 aca ctt cag tca ggt act act gtt gta agaggg ccc ggg ttt acg gga 1488 Thr Leu Gln Ser Gly Thr Thr Val Val Arg GlyPro Gly Phe Thr Gly 485 490 495 gga gat att ctt cga cga aca agt gga ggacca ttt gct tat act att 1536 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly ProPhe Ala Tyr Thr Ile 500 505 510 gtt aat ata aat ggg caa tta ccc caa aggtat cgt gca aga ata cgc 1584 Val Asn Ile Asn Gly Gln Leu Pro Gln Arg TyrArg Ala Arg Ile Arg 515 520 525 tat gcc tct act aca aat cta aga att tacgta acg gtt gca ggt gaa 1632 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr ValThr Val Ala Gly Glu 530 535 540 cgg att ttt gct ggt caa ttt aac aaa acaatg gat acc ggt gac cca 1680 Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr MetAsp Thr Gly Asp Pro 545 550 555 560 tta aca ttc caa tct ttt agt tac gcaact att aat aca gct ttt aca 1728 Leu Thr Phe Gln Ser Phe Ser Tyr Ala ThrIle Asn Thr Ala Phe Thr 565 570 575 ttc cca atg agc cag agt agt ttc acagta ggt gct gat act ttt agt 1776 Phe Pro Met Ser Gln Ser Ser Phe Thr ValGly Ala Asp Thr Phe Ser 580 585 590 tca ggg aat gaa gtt tat ata gac agattt gaa ttg att cca gtt act 1824 Ser Gly Asn Glu Val Tyr Ile Asp Arg PheGlu Leu Ile Pro Val Thr 595 600 605 gca aca ttt gaa gca gaa tat gat ttagaa aga gca caa aag gcg gtg 1872 Ala Thr Phe Glu Ala Glu Tyr Asp Leu GluArg Ala Gln Lys Ala Val 610 615 620 aat gcg ctg ttt act tct ata aac caaata ggg ata aaa aca gat gtg 1920 Asn Ala Leu Phe Thr Ser Ile Asn Gln IleGly Ile Lys Thr Asp Val 625 630 635 640 acg gat tat cat att gat caa gtatcc aat tta gtg gat tgt tta tca 1968 Thr Asp Tyr His Ile Asp Gln Val SerAsn Leu Val Asp Cys Leu Ser 645 650 655 gat gaa ttt tgt ctg gat gaa aagcga gaa ttg tcc gag aaa gtc aaa 2016 Asp Glu Phe Cys Leu Asp Glu Lys ArgGlu Leu Ser Glu Lys Val Lys 660 665 670 cat gcg aag cga ctc agt gat gagcgg aat tta ctt caa gat cca aac 2064 His Ala Lys Arg Leu Ser Asp Glu ArgAsn Leu Leu Gln Asp Pro Asn 675 680 685 ttc aaa ggc atc aat agg caa ctagac cgt ggt tgg aga gga agt acg 2112 Phe Lys Gly Ile Asn Arg Gln Leu AspArg Gly Trp Arg Gly Ser Thr 690 695 700 gat att acc atc caa aga gga gatgac gta ttc aaa gaa aat tat gtc 2160 Asp Ile Thr Ile Gln Arg Gly Asp AspVal Phe Lys Glu Asn Tyr Val 705 710 715 720 aca cta cca ggt acc ttt gatgag tgc tat cca aca tat ttg tat caa 2208 Thr Leu Pro Gly Thr Phe Asp GluCys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 aaa atc gat gaa tca aaa ttaaaa gcc ttt acc cgt tat caa tta aga 2256 Lys Ile Asp Glu Ser Lys Leu LysAla Phe Thr Arg Tyr Gln Leu Arg 740 745 750 ggg tat atc gaa gat agt caagac tta gaa atc tat tta att cgc tac 2304 Gly Tyr Ile Glu Asp Ser Gln AspLeu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 aat gca aaa cat gaa aca gtaaat gtg cca ggt acg ggt tcc tta tgg 2352 Asn Ala Lys His Glu Thr Val AsnVal Pro Gly Thr Gly Ser Leu Trp 770 775 780 ccg ctt tca gcc caa agt ccaatc gga aag tgt gga gag ccg aat cga 2400 Pro Leu Ser Ala Gln Ser Pro IleGly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 tgc gcg cca cac ctt gaatgg aat cct gac tta gat tgt tcg tgt agg 2448 Cys Ala Pro His Leu Glu TrpAsn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 gat gga gaa aag tgt gcccat cat tcg cat cat ttc tcc tta gac att 2496 Asp Gly Glu Lys Cys Ala HisHis Ser His His Phe Ser Leu Asp Ile 820 825 830 gat gta gga tgt aca gactta aat gag gac cta ggt gta tgg gtg atc 2544 Asp Val Gly Cys Thr Asp LeuAsn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 ttt aag att aag acg caagat ggg cac gca aga cta ggg aat cta gag 2592 Phe Lys Ile Lys Thr Gln AspGly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 ttt ctc gaa gag aaa ccatta gta gga gaa gcg cta gct cgt gtg aaa 2640 Phe Leu Glu Glu Lys Pro LeuVal Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880 aga gcg gag aaa aaatgg aga gac aaa cgt gaa aaa ttg gaa tgg gaa 2688 Arg Ala Glu Lys Lys TrpArg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895 aca aat atc gtt tataaa gag gca aaa gaa tct gta gat gct tta ttt 2736 Thr Asn Ile Val Tyr LysGlu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910 gta aac tct caa tatgat caa tta caa gcg gat acg aat att gcc atg 2784 Val Asn Ser Gln Tyr AspGln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925 att cat gcg gca gataaa cgt gtt cat agc att cga gaa gct tat ctg 2832 Ile His Ala Ala Asp LysArg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940 cct gag ctg tct gtgatt ccg ggt gtc aat gcg gct att ttt gaa gaa 2880 Pro Glu Leu Ser Val IlePro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 tta gaa ggg cgtatt ttc act gca ttc tcc cta tat gat gcg aga aat 2928 Leu Glu Gly Arg IlePhe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 gtc att aaa aatggt gat ttt aat aat ggc tta tcc tgc tgg aac gtg 2976 Val Ile Lys Asn GlyAsp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 aaa ggg cat gtagat gta gaa gaa caa aac aac caa cgt tcg gtc ctt 3024 Lys Gly His Val AspVal Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 gtt gtt ccggaa tgg gaa gca gaa gtg tca caa gaa gtt cgt gtc 3069 Val Val Pro Glu TrpGlu Ala Glu Val Ser Gln Glu Val Arg Val 1010 1015 1020 tgt ccg ggt cgtggc tat atc ctt cgt gtc aca gcg tac aag gag 3114 Cys Pro Gly Arg Gly TyrIle Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 gga tat gga gaa ggttgc gta acc att cat gag atc gag aac aat 3159 Gly Tyr Gly Glu Gly Cys ValThr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 aca gac gaa ctg aag tttagc aac tgc gta gaa gag gaa atc tat 3204 Thr Asp Glu Leu Lys Phe Ser AsnCys Val Glu Glu Glu Ile Tyr 1055 1060 1065 cca aat aac acg gta acg tgtaat gat tat act gta aat caa gaa 3249 Pro Asn Asn Thr Val Thr Cys Asn AspTyr Thr Val Asn Gln Glu 1070 1075 1080 gaa tac gga ggt gcg tac act tctcgt aat cga gga tat aac gaa 3294 Glu Tyr Gly Gly Ala Tyr Thr Ser Arg AsnArg Gly Tyr Asn Glu 1085 1090 1095 gct cct tcc gta cca gct gat tat gcgtca gtc tat gaa gaa aaa 3339 Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser ValTyr Glu Glu Lys 1100 1105 1110 tcg tat aca gat gga cga aga gag aat ccttgt gaa ttt aac aga 3384 Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys GluPhe Asn Arg 1115 1120 1125 ggg tat agg gat tac acg cca cta cca gtt ggttat gtg aca aaa 3429 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr ValThr Lys 1130 1135 1140 gaa tta gaa tac ttc cca gaa acc gat aag gta tggatt gag att 3474 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile GluIle 1145 1150 1155 gga gaa acg gaa gga aca ttt atc gtg gac agc gtg gaatta ctc 3519 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu1160 1165 1170 ctt atg gag gaa 3531 Leu Met Glu Glu 1175 14 1177 PRTArtificial sequence Hybrid Delta-Endotoxin 14 Met Asp Asn Asn Pro AsnIle Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu ValGlu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro GlyAla Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe GlyPro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu IleAsn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg LeuGlu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe ArgGlu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu MetArg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 IlePro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr AlaVal 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro AspSer Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr AspSer Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg GluIle Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe ArgGly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser Pro His LeuMet Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Ala His ArgGly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly ThrMet Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 Gln Leu GlyGln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg ProPhe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 GlyThr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser IleIle 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr ProThr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu ValLys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr Thr Val Val Arg GlyPro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Gly GlyPro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn Gly Gln Leu Pro GlnArg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu ArgIle Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile Phe Ala Gly Gln PheAsn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 Leu Thr Phe Gln SerPhe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 Phe Pro Met SerGln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 Ser Gly AsnGlu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 Ala ThrPhe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AsnAla Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp ProAsn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg GlySer Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys GluAsn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr ProThr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser Lys Leu Lys Ala PheThr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu Asp Ser Gln Asp LeuGlu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys His Glu Thr Val AsnVal Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu Ser Ala Gln Ser ProIle Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 Cys Ala Pro His LeuGlu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 Asp Gly Glu LysCys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 Asp Val GlyCys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 Phe LysIle Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 PheLeu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile AlaMet 915 920 925 Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu AlaTyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala IlePhe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser LeuTyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly Asp Phe Asn Asn GlyLeu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val Asp Val Glu Glu GlnAsn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val Pro Glu Trp Glu AlaGlu Val Ser Gln Glu Val Arg Val 1010 1015 1020 Cys Pro Gly Arg Gly TyrIle Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 Gly Tyr Gly Glu GlyCys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 Thr Asp Glu LeuLys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 Pro Asn AsnThr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080 Glu TyrGly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu 1085 1090 1095 AlaPro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys 1100 1105 1110Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg 1115 11201125 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys 11301135 1140 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile1145 1150 1155 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu LeuLeu 1160 1165 1170 Leu Met Glu Glu 1175 15 20 DNA Artificial sequenceSynthetic Oligonucleotide 15 tatccaattc gaacgtcatc 20 16 20 DNAArtificial sequence Synthetic Oligonucleotide 16 tttagtcatc gattaaatca20 17 20 DNA Artificial sequence Synthetic Oligonucleotide 17 ataataagagctccaatgtt 20 18 20 DNA Artificial sequence Synthetic Oligonucleotide 18tacatcgtag tgcaactctt 20 19 20 DNA Artificial sequence SyntheticOligonucleotide 19 tcatggagag ctcctatgtt 20 20 20 DNA Artificialsequence Synthetic Oligonucleotide 20 ttaacaagag ctcctatgtt 20 21 20 DNAArtificial sequence Synthetic Oligonucleotide 21 actaccaggt acctttgatg20 22 20 DNA Artificial sequence Synthetic Oligonucleotide 22 actaccgggtacctttgata 20 23 18 DNA Artificial sequence Synthetic Oligonucleotide 23atttgagtaa tactatcc 18 24 19 DNA Artificial sequence SyntheticOligonucleotide 24 attactcaaa taccattgg 19 25 3534 DNA Artificialsequence Hybrid Delta-Endotoxin 25 atg gat aac aat ccg aac atc aat gaatgc att cct tat aat tgt tta 48 Met Asp Asn Asn Pro Asn Ile Asn Glu CysIle Pro Tyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gta gaa gta tta ggtgga gaa aga ata gaa act ggt 96 Ser Asn Pro Glu Val Glu Val Leu Gly GlyGlu Arg Ile Glu Thr Gly 20 25 30 tac acc cca atc gat att tcc ttg tcg ctaacg caa ttt ctt ttg agt 144 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu ThrGln Phe Leu Leu Ser 35 40 45 gaa ttt gtt ccc ggt gct gga ttt gtg tta ggacta gtt gat ata ata 192 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly LeuVal Asp Ile Ile 50 55 60 tgg gga att ttt ggt ccc tct caa tgg gac gca tttctt gta caa att 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe LeuVal Gln Ile 65 70 75 80 gaa cag tta att aac caa aga ata gaa gaa ttc gctagg aac caa gcc 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala ArgAsn Gln Ala 85 90 95 att tct aga tta gaa gga cta agc aat ctt tat caa atttac gca gaa 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile TyrAla Glu 100 105 110 tct ttt aga gag tgg gaa gca gat cct act aat cca gcatta aga gaa 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala LeuArg Glu 115 120 125 gag atg cgt att caa ttc aat gac atg aac agt gcc cttaca acc gct 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu ThrThr Ala 130 135 140 att cct ctt ttt gca gtt caa aat tat caa gtt cct ctttta tca gta 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu LeuSer Val 145 150 155 160 tat gtt caa gct gca aat tta cat tta tca gtt ttgaga gat gtt tca 528 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu ArgAsp Val Ser 165 170 175 gtg ttt gga caa agg tgg gga ttt gat gcc gcg actatc aat agt cgt 576 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr IleAsn Ser Arg 180 185 190 tat aat gat tta act agg ctt att ggc aac tat acagat cat gct gta 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr AspHis Ala Val 195 200 205 cgc tgg tac aat acg gga tta gag cgt gta tgg ggaccg gat tct aga 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly ProAsp Ser Arg 210 215 220 gat tgg ata aga tat aat caa ttt aga aga gaa ttaaca cta act gta 720 Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 tta gat atc gtt tct cta ttt ccg aac tat gatagt aga acg tat cca 768 Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp SerArg Thr Tyr Pro 245 250 255 att cga aca gtt tcc caa tta aca aga gaa atttat aca aac cca gta 816 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile TyrThr Asn Pro Val 260 265 270 tta gaa aat ttt gat ggt agt ttt cga ggc tcggct cag ggc ata gaa 864 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser AlaGln Gly Ile Glu 275 280 285 aga agt att agg agt cca cat ttg atg gat atactt aac agt ata acc 912 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile LeuAsn Ser Ile Thr 290 295 300 atc tat acg gat gct cat agg ggt tat tat tattgg tca ggg cat caa 960 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr TrpSer Gly His Gln 305 310 315 320 ata atg gct tct cct gta ggg ttt tcg gggcca gaa ttc act ttt ccg 1008 Ile Met Ala Ser Pro Val Gly Phe Ser Gly ProGlu Phe Thr Phe Pro 325 330 335 cta tat gga act atg gga aat gca gct ccacaa caa cgt att gtt gct 1056 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro GlnGln Arg Ile Val Ala 340 345 350 caa cta ggt cag ggc gtg tat aga aca ttatcg tcc act tta tat aga 1104 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu SerSer Thr Leu Tyr Arg 355 360 365 aga cct ttt aat ata ggg ata aat aat caacaa cta tct gtt ctt gac 1152 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln GlnLeu Ser Val Leu Asp 370 375 380 ggg aca gaa ttt gct tat gga acc tcc tcaaat ttg cca tcc gct gta 1200 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser AsnLeu Pro Ser Ala Val 385 390 395 400 tac aga aaa agc gga acg gta gat tcgctg gat gaa ata ccg cca cag 1248 Tyr Arg Lys Ser Gly Thr Val Asp Ser LeuAsp Glu Ile Pro Pro Gln 405 410 415 aat aac aac gtg cca cct agg caa ggattt agt cat cga tta agc cat 1296 Asn Asn Asn Val Pro Pro Arg Gln Gly PheSer His Arg Leu Ser His 420 425 430 gtt tca atg ttt cgt tca ggc ttt agtaat agt agt gta agt ata ata 1344 Val Ser Met Phe Arg Ser Gly Phe Ser AsnSer Ser Val Ser Ile Ile 435 440 445 aga gct cca atg ttt tct tgg acg caccgt agt gca acc cct aca aat 1392 Arg Ala Pro Met Phe Ser Trp Thr His ArgSer Ala Thr Pro Thr Asn 450 455 460 aca att gat ccg gag agg att act caaata cca ttg gta aaa gca cat 1440 Thr Ile Asp Pro Glu Arg Ile Thr Gln IlePro Leu Val Lys Ala His 465 470 475 480 aca ctt cag tca ggt act act gttgta aga ggg ccc ggg ttt acg gga 1488 Thr Leu Gln Ser Gly Thr Thr Val ValArg Gly Pro Gly Phe Thr Gly 485 490 495 gga gat att ctt cga cga aca agtgga gga cca ttt gct tat act att 1536 Gly Asp Ile Leu Arg Arg Thr Ser GlyGly Pro Phe Ala Tyr Thr Ile 500 505 510 gtt aat ata aat ggg caa tta ccccaa agg tat cgt gca aga ata cgc 1584 Val Asn Ile Asn Gly Gln Leu Pro GlnArg Tyr Arg Ala Arg Ile Arg 515 520 525 tat gcc tct act aca aat cta agaatt tac gta acg gtt gca ggt gaa 1632 Tyr Ala Ser Thr Thr Asn Leu Arg IleTyr Val Thr Val Ala Gly Glu 530 535 540 cgg att ttt gct ggt caa ttt aacaaa aca atg gat acc ggt gac cca 1680 Arg Ile Phe Ala Gly Gln Phe Asn LysThr Met Asp Thr Gly Asp Pro 545 550 555 560 tta aca ttc caa tct ttt agttac gca act att aat aca gct ttt aca 1728 Leu Thr Phe Gln Ser Phe Ser TyrAla Thr Ile Asn Thr Ala Phe Thr 565 570 575 ttc cca atg agc cag agt agtttc aca gta ggt gct gat act ttt agt 1776 Phe Pro Met Ser Gln Ser Ser PheThr Val Gly Ala Asp Thr Phe Ser 580 585 590 tca ggg aat gaa gtt tat atagac aga ttt gaa ttg att cca gtt act 1824 Ser Gly Asn Glu Val Tyr Ile AspArg Phe Glu Leu Ile Pro Val Thr 595 600 605 gca aca ttt gaa gca gaa tatgat tta gaa aga gca caa aag gcg gtg 1872 Ala Thr Phe Glu Ala Glu Tyr AspLeu Glu Arg Ala Gln Lys Ala Val 610 615 620 aat gcg ctg ttt act tct ataaac caa ata ggg ata aaa aca gat gtg 1920 Asn Ala Leu Phe Thr Ser Ile AsnGln Ile Gly Ile Lys Thr Asp Val 625 630 635 640 acg gat tat cat att gatcaa gta tcc aat tta gtg gat tgt tta tca 1968 Thr Asp Tyr His Ile Asp GlnVal Ser Asn Leu Val Asp Cys Leu Ser 645 650 655 gat gaa ttt tgt ctg gatgaa aag cga gaa ttg tcc gag aaa gtc aaa 2016 Asp Glu Phe Cys Leu Asp GluLys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 cat gcg aag cga ctc agtgat gag cgg aat tta ctt caa gat cca aac 2064 His Ala Lys Arg Leu Ser AspGlu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685 ttc aaa ggc atc aat aggcaa cta gac cgt ggt tgg aga gga agt acg 2112 Phe Lys Gly Ile Asn Arg GlnLeu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700 gat att acc atc caa agagga gat gac gta ttc aaa gaa aat tat gtc 2160 Asp Ile Thr Ile Gln Arg GlyAsp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 aca cta cca ggt accttt gat gag tgc tat cca aca tat ttg tat caa 2208 Thr Leu Pro Gly Thr PheAsp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 aaa atc gat gaa tcaaaa tta aaa gcc ttt acc cgt tat caa tta aga 2256 Lys Ile Asp Glu Ser LysLeu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 ggg tat atc gaa gatagt caa gac tta gaa atc tat tta att cgc tac 2304 Gly Tyr Ile Glu Asp SerGln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 aat gca aaa cat gaaaca gta aat gtg cca ggt acg ggt tcc tta tgg 2352 Asn Ala Lys His Glu ThrVal Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 ccg ctt tca gcc caaagt cca atc gga aag tgt gga gag ccg aat cga 2400 Pro Leu Ser Ala Gln SerPro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 tgc gcg cca cacctt gaa tgg aat cct gac tta gat tgt tcg tgt agg 2448 Cys Ala Pro His LeuGlu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 gat gga gaa aagtgt gcc cat cat tcg cat cat ttc tcc tta gac att 2496 Asp Gly Glu Lys CysAla His His Ser His His Phe Ser Leu Asp Ile 820 825 830 gat gta gga tgtaca gac tta aat gag gac cta ggt gta tgg gtg atc 2544 Asp Val Gly Cys ThrAsp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 ttt aag att aagacg caa gat ggg cac gca aga cta ggg aat cta gag 2592 Phe Lys Ile Lys ThrGln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 ttt ctc gaa gagaaa cca tta gta gga gaa gcg cta gct cgt gtg aaa 2640 Phe Leu Glu Glu LysPro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880 aga gcg gagaaa aaa tgg aga gac aaa cgt gaa aaa ttg gaa tgg gaa 2688 Arg Ala Glu LysLys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895 aca aat atcgtt tat aaa gag gca aaa gaa tct gta gat gct tta ttt 2736 Thr Asn Ile ValTyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910 gta aac tctcaa tat gat caa tta caa gcg gat acg aat att gcc atg 2784 Val Asn Ser GlnTyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925 att cat gcggca gat aaa cgt gtt cat agc att cga gaa gct tat ctg 2832 Ile His Ala AlaAsp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940 cct gag ctgtct gtg att ccg ggt gtc aat gcg gct att ttt gaa gaa 2880 Pro Glu Leu SerVal Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 tta gaaggg cgt att ttc act gca ttc tcc cta tat gat gcg aga aat 2928 Leu Glu GlyArg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 gtc attaaa aat ggt gat ttt aat aat ggc tta tcc tgc tgg aac gtg 2976 Val Ile LysAsn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 aaa gggcat gta gat gta gaa gaa caa aac aac caa cgt tcg gtc ctt 3024 Lys Gly HisVal Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 gttgtt ccg gaa tgg gaa gca gaa gtg tca caa gaa gtt cgt gtc 3069 Val Val ProGlu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val 1010 1015 1020 tgt ccgggt cgt ggc tat atc ctt cgt gtc aca gcg tac aag gag 3114 Cys Pro Gly ArgGly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 gga tat ggagaa ggt tgc gta acc att cat gag atc gag aac aat 3159 Gly Tyr Gly Glu GlyCys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 aca gac gaa ctgaag ttt agc aac tgc gta gaa gag gaa atc tat 3204 Thr Asp Glu Leu Lys PheSer Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 cca aat aac acg gtaacg tgt aat gat tat act gta aat caa gaa 3249 Pro Asn Asn Thr Val Thr CysAsn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080 gaa tac gga ggt gcg tacact tct cgt aat cga gga tat aac gaa 3294 Glu Tyr Gly Gly Ala Tyr Thr SerArg Asn Arg Gly Tyr Asn Glu 1085 1090 1095 gct cct tcc gta cca gct gattat gcg tca gtc tat gaa gaa aaa 3339 Ala Pro Ser Val Pro Ala Asp Tyr AlaSer Val Tyr Glu Glu Lys 1100 1105 1110 tcg tat aca gat gga cga aga gagaat cct tgt gaa ttt aac aga 3384 Ser Tyr Thr Asp Gly Arg Arg Glu Asn ProCys Glu Phe Asn Arg 1115 1120 1125 ggg tat agg gat tac acg cca cta ccagtt ggt tat gtg aca aaa 3429 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val GlyTyr Val Thr Lys 1130 1135 1140 gaa tta gaa tac ttc cca gaa acc gat aaggta tgg att gag att 3474 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val TrpIle Glu Ile 1145 1150 1155 gga gaa acg gaa gga aca ttt atc gtg gac agcgtg gaa tta ctc 3519 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val GluLeu Leu 1160 1165 1170 ctt atg gag gaa tag 3534 Leu Met Glu Glu 1175 261177 PRT Artificial sequence Hybrid Delta-Endotoxin 26 Met Asp Asn AsnPro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn ProGlu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr ProIle Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe ValPro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly IlePhe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu GlnLeu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile SerArg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 SerPhe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp ValSer 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile AsnSer Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr AspHis Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp GlyPro Asp Ser Arg 210 215 220 Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg GluLeu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ser Leu Phe Pro AsnTyr Asp Ser Arg Thr Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu ThrArg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly SerPhe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser ProHis Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp AlaHis Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met AlaSer Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu TyrGly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 GlnLeu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro ProGln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg LeuSer His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser ValSer Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser AlaThr Pro Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile ProLeu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr Thr Val ValArg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr SerGly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn Gly Gln LeuPro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr AsnLeu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile Phe Ala GlyGln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 Leu Thr PheGln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 Phe ProMet Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 SerGly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys LeuSer 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu LysVal Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu GlnAsp Pro Asn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly TrpArg Gly Ser Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val PheLys Glu Asn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe Asp Glu CysTyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser Lys Leu LysAla Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu Asp Ser GlnAsp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys His Glu ThrVal Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu Ser Ala GlnSer Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 Cys Ala ProHis Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 Asp GlyGlu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 AspVal Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu TrpGlu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp AlaLeu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr AsnIle Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val His Ser Ile ArgGlu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn AlaAla Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe Thr Ala PheSer Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly Asp Phe AsnAsn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val Asp Val GluGlu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val Pro Glu TrpGlu Ala Glu Val Ser Gln Glu Val Arg Val 1010 1015 1020 Cys Pro Gly ArgGly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 Gly Tyr GlyGlu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 Thr AspGlu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 ProAsn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu 1085 10901095 Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys 11001105 1110 Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg1115 1120 1125 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val ThrLys 1130 1135 1140 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp IleGlu Ile 1145 1150 1155 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser ValGlu Leu Leu 1160 1165 1170 Leu Met Glu Glu 1175 27 3534 DNA Artificialsequence Hybrid Delta-Endotoxin 27 atg gat aac aat ccg aac atc aat gaatgc att cct tat aat tgt tta 48 Met Asp Asn Asn Pro Asn Ile Asn Glu CysIle Pro Tyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gta gaa gta tta ggtgga gaa aga ata gaa act ggt 96 Ser Asn Pro Glu Val Glu Val Leu Gly GlyGlu Arg Ile Glu Thr Gly 20 25 30 tac acc cca atc gat att tcc ttg tcg ctaacg caa ttt ctt ttg agt 144 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu ThrGln Phe Leu Leu Ser 35 40 45 gaa ttt gtt ccc ggt gct gga ttt gtg tta ggacta gtt gat ata ata 192 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly LeuVal Asp Ile Ile 50 55 60 tgg gga att ttt ggt ccc tct caa tgg gac gca tttctt gta caa att 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe LeuVal Gln Ile 65 70 75 80 gaa cag tta att aac caa aga ata gaa gaa ttc gctagg aac caa gcc 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala ArgAsn Gln Ala 85 90 95 att tct aga tta gaa gga cta agc aat ctt tat caa atttac gca gaa 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile TyrAla Glu 100 105 110 tct ttt aga gag tgg gaa gca gat cct act aat cca gcatta aga gaa 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala LeuArg Glu 115 120 125 gag atg cgt att caa ttc aat gac atg aac agt gcc cttaca acc gct 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu ThrThr Ala 130 135 140 att cct ctt ttt gca gtt caa aat tat caa gtt cct ctttta tca gta 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu LeuSer Val 145 150 155 160 tat gtt caa gct gca aat tta cat tta tca gtt ttgaga gat gtt tca 528 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu ArgAsp Val Ser 165 170 175 gtg ttt gga caa agg tgg gga ttt gat gcc gcg actatc aat agt cgt 576 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr IleAsn Ser Arg 180 185 190 tat aat gat tta act agg ctt att ggc aac tat acagat tat gct gta 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr AspTyr Ala Val 195 200 205 cgc tgg tac aat acg gga tta gaa cgt gta tgg ggaccg gat tct aga 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly ProAsp Ser Arg 210 215 220 gat tgg gta agg tat aat caa ttt aga aga gaa ttaaca cta act gta 720 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 tta gat atc gtt gct ctg ttc ccg aat tat gatagt aga aga tat cca 768 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp SerArg Arg Tyr Pro 245 250 255 att cga aca gtt tcc caa tta aca aga gaa atttat aca aac cca gta 816 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile TyrThr Asn Pro Val 260 265 270 tta gaa aat ttt gat ggt agt ttt cga ggc tcggct cag ggc ata gaa 864 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser AlaGln Gly Ile Glu 275 280 285 aga agt att agg agt cca cat ttg atg gat atactt aac agt ata acc 912 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile LeuAsn Ser Ile Thr 290 295 300 atc tat acg gat gct cat agg ggt tat tat tattgg tca ggg cat caa 960 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr TrpSer Gly His Gln 305 310 315 320 ata atg gct tct cct gta ggg ttt tcg gggcca gaa ttc act ttt ccg 1008 Ile Met Ala Ser Pro Val Gly Phe Ser Gly ProGlu Phe Thr Phe Pro 325 330 335 cta tat gga act atg gga aat gca gct ccacaa caa cgt att gtt gct 1056 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro GlnGln Arg Ile Val Ala 340 345 350 caa cta ggt cag ggc gtg tat aga aca ttatcg tcc act tta tat aga 1104 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu SerSer Thr Leu Tyr Arg 355 360 365 aga cct ttt aat ata ggg ata aat aat caacaa cta tct gtt ctt gac 1152 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln GlnLeu Ser Val Leu Asp 370 375 380 ggg aca gaa ttt gct tat gga acc tcc tcaaat ttg cca tcc gct gta 1200 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser AsnLeu Pro Ser Ala Val 385 390 395 400 tac aga aaa agc gga acg gta gat tcgctg gat gaa ata ccg cca cag 1248 Tyr Arg Lys Ser Gly Thr Val Asp Ser LeuAsp Glu Ile Pro Pro Gln 405 410 415 aat aac aac gtg cca cct agg caa ggattt agt cat cga tta agc cat 1296 Asn Asn Asn Val Pro Pro Arg Gln Gly PheSer His Arg Leu Ser His 420 425 430 gtt tca atg ttt cgt tca ggc ttt agtaat agt agt gta agt ata ata 1344 Val Ser Met Phe Arg Ser Gly Phe Ser AsnSer Ser Val Ser Ile Ile 435 440 445 aga gct cct atg ttc tct tgg ata catcgt agt gct gaa ttt aat aat 1392 Arg Ala Pro Met Phe Ser Trp Ile His ArgSer Ala Glu Phe Asn Asn 450 455 460 ata att gca tcg gat agt att act caaata cca ttg gta aaa gca cat 1440 Ile Ile Ala Ser Asp Ser Ile Thr Gln IlePro Leu Val Lys Ala His 465 470 475 480 aca ctt cag tca ggt act act gttgta aga ggg ccc ggg ttt acg gga 1488 Thr Leu Gln Ser Gly Thr Thr Val ValArg Gly Pro Gly Phe Thr Gly 485 490 495 gga gat att ctt cga cga aca agtgga gga cca ttt gct tat act att 1536 Gly Asp Ile Leu Arg Arg Thr Ser GlyGly Pro Phe Ala Tyr Thr Ile 500 505 510 gtt aat ata aat ggg caa tta ccccaa agg tat cgt gca aga ata cgc 1584 Val Asn Ile Asn Gly Gln Leu Pro GlnArg Tyr Arg Ala Arg Ile Arg 515 520 525 tat gcc tct act aca aat cta agaatt tac gta acg gtt gca ggt gaa 1632 Tyr Ala Ser Thr Thr Asn Leu Arg IleTyr Val Thr Val Ala Gly Glu 530 535 540 cgg att ttt gct ggt caa ttt aacaaa aca atg gat acc ggt gac cca 1680 Arg Ile Phe Ala Gly Gln Phe Asn LysThr Met Asp Thr Gly Asp Pro 545 550 555 560 tta aca ttc caa tct ttt agttac gca act att aat aca gct ttt aca 1728 Leu Thr Phe Gln Ser Phe Ser TyrAla Thr Ile Asn Thr Ala Phe Thr 565 570 575 ttc cca atg agc cag agt agtttc aca gta ggt gct gat act ttt agt 1776 Phe Pro Met Ser Gln Ser Ser PheThr Val Gly Ala Asp Thr Phe Ser 580 585 590 tca ggg aat gaa gtt tat atagac aga ttt gaa ttg att cca gtt act 1824 Ser Gly Asn Glu Val Tyr Ile AspArg Phe Glu Leu Ile Pro Val Thr 595 600 605 gca aca ttt gaa gca gaa tatgat tta gaa aga gca caa aag gcg gtg 1872 Ala Thr Phe Glu Ala Glu Tyr AspLeu Glu Arg Ala Gln Lys Ala Val 610 615 620 aat gcg ctg ttt act tct ataaac caa ata ggg ata aaa aca gat gtg 1920 Asn Ala Leu Phe Thr Ser Ile AsnGln Ile Gly Ile Lys Thr Asp Val 625 630 635 640 acg gat tat cat att gatcaa gta tcc aat tta gtg gat tgt tta tca 1968 Thr Asp Tyr His Ile Asp GlnVal Ser Asn Leu Val Asp Cys Leu Ser 645 650 655 gat gaa ttt tgt ctg gatgaa aag cga gaa ttg tcc gag aaa gtc aaa 2016 Asp Glu Phe Cys Leu Asp GluLys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 cat gcg aag cga ctc agtgat gag cgg aat tta ctt caa gat cca aac 2064 His Ala Lys Arg Leu Ser AspGlu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685 ttc aaa ggc atc aat aggcaa cta gac cgt ggt tgg aga gga agt acg 2112 Phe Lys Gly Ile Asn Arg GlnLeu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700 gat att acc atc caa agagga gat gac gta ttc aaa gaa aat tat gtc 2160 Asp Ile Thr Ile Gln Arg GlyAsp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 aca cta cca ggt accttt gat gag tgc tat cca aca tat ttg tat caa 2208 Thr Leu Pro Gly Thr PheAsp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 aaa atc gat gaa tcaaaa tta aaa gcc ttt acc cgt tat caa tta aga 2256 Lys Ile Asp Glu Ser LysLeu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 ggg tat atc gaa gatagt caa gac tta gaa atc tat tta att cgc tac 2304 Gly Tyr Ile Glu Asp SerGln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 aat gca aaa cat gaaaca gta aat gtg cca ggt acg ggt tcc tta tgg 2352 Asn Ala Lys His Glu ThrVal Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 ccg ctt tca gcc caaagt cca atc gga aag tgt gga gag ccg aat cga 2400 Pro Leu Ser Ala Gln SerPro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 tgc gcg cca cacctt gaa tgg aat cct gac tta gat tgt tcg tgt agg 2448 Cys Ala Pro His LeuGlu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 gat gga gaa aagtgt gcc cat cat tcg cat cat ttc tcc tta gac att 2496 Asp Gly Glu Lys CysAla His His Ser His His Phe Ser Leu Asp Ile 820 825 830 gat gta gga tgtaca gac tta aat gag gac cta ggt gta tgg gtg atc 2544 Asp Val Gly Cys ThrAsp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 ttt aag att aagacg caa gat ggg cac gca aga cta ggg aat cta gag 2592 Phe Lys Ile Lys ThrGln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 ttt ctc gaa gagaaa cca tta gta gga gaa gcg cta gct cgt gtg aaa 2640 Phe Leu Glu Glu LysPro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880 aga gcg gagaaa aaa tgg aga gac aaa cgt gaa aaa ttg gaa tgg gaa 2688 Arg Ala Glu LysLys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895 aca aat atcgtt tat aaa gag gca aaa gaa tct gta gat gct tta ttt 2736 Thr Asn Ile ValTyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910 gta aac tctcaa tat gat caa tta caa gcg gat acg aat att gcc atg 2784 Val Asn Ser GlnTyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925 att cat gcggca gat aaa cgt gtt cat agc att cga gaa gct tat ctg 2832 Ile His Ala AlaAsp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940 cct gag ctgtct gtg att ccg ggt gtc aat gcg gct att ttt gaa gaa 2880 Pro Glu Leu SerVal Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 tta gaaggg cgt att ttc act gca ttc tcc cta tat gat gcg aga aat 2928 Leu Glu GlyArg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 gtc attaaa aat ggt gat ttt aat aat ggc tta tcc tgc tgg aac gtg 2976 Val Ile LysAsn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 aaa gggcat gta gat gta gaa gaa caa aac aac caa cgt tcg gtc ctt 3024 Lys Gly HisVal Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 gttgtt ccg gaa tgg gaa gca gaa gtg tca caa gaa gtt cgt gtc 3069 Val Val ProGlu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val 1010 1015 1020 tgt ccgggt cgt ggc tat atc ctt cgt gtc aca gcg tac aag gag 3114 Cys Pro Gly ArgGly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 gga tat ggagaa ggt tgc gta acc att cat gag atc gag aac aat 3159 Gly Tyr Gly Glu GlyCys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 aca gac gaa ctgaag ttt agc aac tgc gta gaa gag gaa atc tat 3204 Thr Asp Glu Leu Lys PheSer Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 cca aat aac acg gtaacg tgt aat gat tat act gta aat caa gaa 3249 Pro Asn Asn Thr Val Thr CysAsn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080 gaa tac gga ggt gcg tacact tct cgt aat cga gga tat aac gaa 3294 Glu Tyr Gly Gly Ala Tyr Thr SerArg Asn Arg Gly Tyr Asn Glu 1085 1090 1095 gct cct tcc gta cca gct gattat gcg tca gtc tat gaa gaa aaa 3339 Ala Pro Ser Val Pro Ala Asp Tyr AlaSer Val Tyr Glu Glu Lys 1100 1105 1110 tcg tat aca gat gga cga aga gagaat cct tgt gaa ttt aac aga 3384 Ser Tyr Thr Asp Gly Arg Arg Glu Asn ProCys Glu Phe Asn Arg 1115 1120 1125 ggg tat agg gat tac acg cca cta ccagtt ggt tat gtg aca aaa 3429 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val GlyTyr Val Thr Lys 1130 1135 1140 gaa tta gaa tac ttc cca gaa acc gat aaggta tgg att gag att 3474 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val TrpIle Glu Ile 1145 1150 1155 gga gaa acg gaa gga aca ttt atc gtg gac agcgtg gaa tta ctc 3519 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val GluLeu Leu 1160 1165 1170 ctt atg gag gaa tag 3534 Leu Met Glu Glu 1175 281177 PRT Artificial sequence Hybrid Delta-Endotoxin 28 Met Asp Asn AsnPro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn ProGlu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr ProIle Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe ValPro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly IlePhe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu GlnLeu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile SerArg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 SerPhe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp ValSer 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile AsnSer Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr AspTyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp GlyPro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg GluLeu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro AsnTyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu ThrArg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly SerPhe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser ProHis Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp AlaHis Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met AlaSer Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu TyrGly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 GlnLeu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro ProGln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg LeuSer His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser ValSer Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser AlaGlu Phe Asn Asn 450 455 460 Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile ProLeu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr Thr Val ValArg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr SerGly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn Gly Gln LeuPro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr AsnLeu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile Phe Ala GlyGln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 Leu Thr PheGln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 Phe ProMet Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 SerGly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys LeuSer 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu LysVal Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu GlnAsp Pro Asn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly TrpArg Gly Ser Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val PheLys Glu Asn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe Asp Glu CysTyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser Lys Leu LysAla Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu Asp Ser GlnAsp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys His Glu ThrVal Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu Ser Ala GlnSer Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 Cys Ala ProHis Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 Asp GlyGlu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 AspVal Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu TrpGlu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp AlaLeu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr AsnIle Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val His Ser Ile ArgGlu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn AlaAla Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe Thr Ala PheSer Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly Asp Phe AsnAsn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val Asp Val GluGlu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val Pro Glu TrpGlu Ala Glu Val Ser Gln Glu Val Arg Val 1010 1015 1020 Cys Pro Gly ArgGly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu 1025 1030 1035 Gly Tyr GlyGlu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn 1040 1045 1050 Thr AspGlu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr 1055 1060 1065 ProAsn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu 1070 1075 1080Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu 1085 10901095 Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys 11001105 1110 Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg1115 1120 1125 Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val ThrLys 1130 1135 1140 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp IleGlu Ile 1145 1150 1155 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser ValGlu Leu Leu 1160 1165 1170 Leu Met Glu Glu 1175 29 3579 DNA Artificialsequence Hybrid Delta-Endotoxin 29 atggataaca atccgaacat caatgaatgcattccttata attgtttaag taaccctgaa 60 gtagaagtat taggtggaga aagaatagaaactggttaca ccccaatcga tatttccttg 120 tcgctaacgc aatttctttt gagtgaatttgttcccggtg ctggatttgt gttaggacta 180 gttgatataa tatggggaat ttttggtccctctcaatggg acgcatttct tgtacaaatt 240 gaacagttaa ttaaccaaag aatagaagaattcgctagga accaagccat ttctagatta 300 gaaggactaa gcaatcttta tcaaatttacgcagaatctt ttagagagtg ggaagcagat 360 cctactaatc cagcattaag agaagagatgcgtattcaat tcaatgacat gaacagtgcc 420 cttacaaccg ctattcctct ttttgcagttcaaaattatc aagttcctct tttatcagta 480 tatgttcaag ctgcaaattt acatttatcagttttgagag atgtttcagt gtttggacaa 540 aggtggggat ttgatgccgc gactatcaatagtcgttata atgatttaac taggcttatt 600 ggcaactata cagattatgc tgtacgctggtacaatacgg gattagaacg tgtatgggga 660 ccggattcta gagattgggt aaggtataatcaatttagaa gagaattaac actaactgta 720 ttagatatcg ttgctctgtt cccgaattatgatagtagaa gatatccaat tcgaacagtt 780 tcccaattaa caagagaaat ttatacaaacccagtattag aaaattttga tggtagtttt 840 cgaggctcgg ctcagggcat agaaagaagtattaggagtc cacatttgat ggatatactt 900 aacagtataa ccatctatac ggatgctcataggggttatt attattggtc agggcatcaa 960 ataatggctt ctcctgtagg gttttcggggccagaattca cttttccgct atatggaact 1020 atgggaaatg cagctccaca acaacgtattgttgctcaac taggtcaggg cgtgtataga 1080 acattatcgt ccactttata tagaagaccttttaatatag ggataaataa tcaacaacta 1140 tctgttcttg acgggacaga atttgcttatggaacctcct caaatttgcc atccgctgta 1200 tacagaaaaa gcggaacggt agattcgctggatgaaatac cgccacagaa taacaacgtg 1260 ccacctaggc aaggatttag tcatcgattaagccatgttt caatgtttcg ttcaggcttt 1320 agtaatagta gtgtaagtat aataagagctcctatgttct cttggataca tcgtagtgca 1380 actcttacaa atacaattga tccagagagaattaatcaaa tacctttagt gaaaggattt 1440 agagtttggg ggggcacctc tgtcattacaggaccaggat ttacaggagg ggatatcctt 1500 cgaagaaata cctttggtga ttttgtatctctacaagtca atattaattc accaattacc 1560 caaagatacc gtttaagatt tcgttacgcttccagtaggg atgcacgagt tatagtatta 1620 acaggagcgg catccacagg agtgggaggccaagttagtg taaatatgcc tcttcagaaa 1680 actatggaaa taggggagaa cttaacatctagaacattta gatataccga ttttagtaat 1740 cctttttcat ttagagctaa tccagatataattgggataa gtgaacaacc tctatttggt 1800 gcaggttcta ttagtagcgg tgaactttatatagataaaa ttgaaattat tctagcagat 1860 gcaacatttg aagcagaatc tgatttagaaagagcacaaa aggcggtgaa tgccctgttt 1920 acttcttcca atcaaatcgg gttaaaaaccgatgtgacgg attatcatat tgatcaagta 1980 tccaatttag tggattgttt atcagatgaattttgtctgg atgaaaagcg agaattgtcc 2040 gagaaagtca aacatgcgaa gcgactcagtgatgagcgga atttacttca agatccaaac 2100 ttcagaggga tcaatagaca accagaccgtggctggagag gaagtacaga tattaccatc 2160 caaggaggag atgacgtatt caaagagaattacgtcacac taccgggtac cgttgatgag 2220 tgctatccaa cgtatttata tcagaaaatagatgagtcga aattaaaagc ttatacccgt 2280 tatgaattaa gagggtatat cgaagatagtcaagacttag aaatctattt gatccgttac 2340 aatgcaaaac acgaaatagt aaatgtgccaggcacgggtt ccttatggcc gctttcagcc 2400 caaagtccaa tcggaaagtg tggagaaccgaatcgatgcg cgccacacct tgaatggaat 2460 cctgatctag attgttcctg cagagacggggaaaaatgtg cacatcattc ccatcatttc 2520 accttggata ttgatgttgg atgtacagacttaaatgagg acttaggtgt atgggtgata 2580 ttcaagatta agacgcaaga tggccatgcaagactaggga atctagagtt tctcgaagag 2640 aaaccattat taggggaagc actagctcgtgtgaaaagag cggagaagaa gtggagagac 2700 aaacgagaga aactgcagtt ggaaacaaatattgtttata aagaggcaaa agaatctgta 2760 gatgctttat ttgtaaactc tcaatatgatagattacaag tggatacgaa catcgcaatg 2820 attcatgcgg cagataaacg cgttcatagaatccgggaag cgtatctgcc agagttgtct 2880 gtgattccag gtgtcaatgc ggccattttcgaagaattag agggacgtat ttttacagcg 2940 tattccttat atgatgcgag aaatgtcattaaaaatggcg atttcaataa tggcttatta 3000 tgctggaacg tgaaaggtca tgtagatgtagaagagcaaa acaaccaccg ttcggtcctt 3060 gttatcccag aatgggaggc agaagtgtcacaagaggttc gtgtctgtcc aggtcgtggc 3120 tatatccttc gtgtcacagc atataaagagggatatggag agggctgcgt aacgatccat 3180 gagatcgaag acaatacaga cgaactgaaattcagcaact gtgtagaaga ggaagtatat 3240 ccaaacaaca cagtaacgtg taataattatactgggactc aagaagaata tgagggtacg 3300 tacacttctc gtaatcaagg atatgacgaagcctatggta ataacccttc cgtaccagct 3360 gattacgctt cagtctatga agaaaaatcgtatacagatg gacgaagaga gaatccttgt 3420 gaatctaaca gaggctatgg ggattacacaccactaccgg ctggttatgt aacaaaggat 3480 ttagagtact tcccagagac cgataaggtatggattgaga tcggagaaac agaaggaaca 3540 ttcatcgtgg atagcgtgga attactccttatggaggaa 3579 30 1193 PRT Artificial sequence Hybrid Delta-Endotoxin 30Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 1015 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 2530 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 4045 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 5560 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 7075 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 8590 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu ArgGlu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu ThrThr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro LeuLeu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser ValLeu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp AlaAla Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile GlyAsn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu GluArg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn GlnPhe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val AlaLeu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr ValSer Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu AsnPhe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg SerIle Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 IleTyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu TyrArg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser ValLeu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu ProSer Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu AspGlu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly PheSer His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe SerAsn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp IleHis Arg Ser Ala Thr Leu Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg IleAsn Gln Ile Pro Leu Val Lys Gly Phe 465 470 475 480 Arg Val Trp Gly GlyThr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile LeuArg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln 500 505 510 Val Asn IleAsn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg 515 520 525 Tyr AlaSer Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala 530 535 540 SerThr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys 545 550 555560 Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr 565570 575 Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly580 585 590 Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser GlyGlu 595 600 605 Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala ThrPhe Glu 610 615 620 Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val AsnAla Leu Phe 625 630 635 640 Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr AspVal Thr Asp Tyr His 645 650 655 Ile Asp Gln Val Ser Asn Leu Val Asp CysLeu Ser Asp Glu Phe Cys 660 665 670 Leu Asp Glu Lys Arg Glu Leu Ser GluLys Val Lys His Ala Lys Arg 675 680 685 Leu Ser Asp Glu Arg Asn Leu LeuGln Asp Pro Asn Phe Arg Gly Ile 690 695 700 Asn Arg Gln Pro Asp Arg GlyTrp Arg Gly Ser Thr Asp Ile Thr Ile 705 710 715 720 Gln Gly Gly Asp AspVal Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly 725 730 735 Thr Val Asp GluCys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu 740 745 750 Ser Lys LeuLys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu 755 760 765 Asp SerGln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His 770 775 780 GluIle Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala 785 790 795800 Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His 805810 815 Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys820 825 830 Cys Ala His His Ser His His Phe Thr Leu Asp Ile Asp Val GlyCys 835 840 845 Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe LysIle Lys 850 855 860 Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu PheLeu Glu Glu 865 870 875 880 Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg ValLys Arg Ala Glu Lys 885 890 895 Lys Trp Arg Asp Lys Arg Glu Lys Leu GlnLeu Glu Thr Asn Ile Val 900 905 910 Tyr Lys Glu Ala Lys Glu Ser Val AspAla Leu Phe Val Asn Ser Gln 915 920 925 Tyr Asp Arg Leu Gln Val Asp ThrAsn Ile Ala Met Ile His Ala Ala 930 935 940 Asp Lys Arg Val His Arg IleArg Glu Ala Tyr Leu Pro Glu Leu Ser 945 950 955 960 Val Ile Pro Gly ValAsn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg 965 970 975 Ile Phe Thr AlaTyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn 980 985 990 Gly Asp PheAsn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val 995 1000 1005 AspVal Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro 1010 1015 1020Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly 1025 10301035 Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly 10401045 1050 Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu1055 1060 1065 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro AsnAsn 1070 1075 1080 Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln Glu GluTyr Glu 1085 1090 1095 Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp GluAla Tyr Gly 1100 1105 1110 Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala SerVal Tyr Glu Glu 1115 1120 1125 Lys Ser Tyr Thr Asp Gly Arg Arg Glu AsnPro Cys Glu Ser Asn 1130 1135 1140 Arg Gly Tyr Gly Asp Tyr Thr Pro LeuPro Ala Gly Tyr Val Thr 1145 1150 1155 Lys Asp Leu Glu Tyr Phe Pro GluThr Asp Lys Val Trp Ile Glu 1160 1165 1170 Ile Gly Glu Thr Glu Gly ThrPhe Ile Val Asp Ser Val Glu Leu 1175 1180 1185 Leu Leu Met Glu Glu 119031 16 DNA Artificial sequence Synthetic Oligonucleotide 31 cgttgctctgttcccg 16 32 20 DNA Artificial sequence Synthetic Oligonucleotide 32tcaaatacca ttggtaaaag 20 33 3534 DNA Artificial sequence HybridDelta-Endotoxin 33 atggataaca atccgaacat caatgaatgc attccttataattgtttaag taaccctgaa 60 gtagaagtat taggtggaga aagaatagaa actggttacaccccaatcga tatttccttg 120 tcgctaacgc aatttctttt gagtgaattt gttcccggtgctggatttgt gttaggacta 180 gttgatataa tatggggaat ttttggtccc tctcaatgggacgcatttct tgtacaaatt 240 gaacagttaa ttaaccaaag aatagaagaa ttcgctaggaaccaagccat ttctagatta 300 gaaggactaa gcaatcttta tcaaatttac gcagaatcttttagagagtg ggaagcagat 360 cctactaatc cagcattaag agaagagatg cgtattcaattcaatgacat gaacagtgcc 420 cttacaaccg ctattcctct ttttgcagtt caaaattatcaagttcctct tttatcagta 480 tatgttcaag ctgcaaattt acatttatca gttttgagagatgtttcagt gtttggacaa 540 aggtggggat ttgatgccgc gactatcaat agtcgttataatgatttaac taggcttatt 600 ggcaactata cagattatgc tgtacgctgg tacaatacgggattagaacg tgtatgggga 660 ccggattcta gagattgggt aaggtataat caatttagaagagaattaac actaactgta 720 ttagatatcg ttgctctgtt cccgaattat gatagtagaagatatccaat tcgaacagtt 780 tcccaattaa caagagaaat ttatacaaac ccagtattagaaaattttga tggtagtttt 840 cgaggctcgg ctcagggcat agaaagaagt attaggagtccacatttgat ggatatactt 900 aacagtataa ccatctatac ggatgctcat aggggttattattattggtc agggcatcaa 960 ataatggctt ctcctgtagg gttttcgggg ccagaattcacttttccgct atatggaact 1020 atgggaaatg cagctccaca acaacgtatt gttgctcaactaggtcaggg cgtgtataga 1080 acattatcgt ccactttata tagaagacct tttaatatagggataaataa tcaacaacta 1140 tctgttcttg acgggacaga atttgcttat ggaacctcctcaaatttgcc atccgctgta 1200 tacagaaaaa gcggaacggt agattcgctg gatgaaataccgccacagaa taacaacgtg 1260 ccacctaggc aaggatttag tcatcgatta agccatgtttcaatgtttcg ttcaggcttt 1320 agtaatagta gtgtaagtat aataagagct cctatgttctcttggataca tcgtagtgct 1380 gaatttaata atataattgc atcggatagt attactcaaataccattggt aaaagcacat 1440 acacttcagt caggtactac tgttgtaaga gggcccgggtttacgggagg agatattctt 1500 cgacgaacaa gtggaggacc atttgcttat actattgttaatataaatgg gcaattaccc 1560 caaaggtatc gtgcaagaat acgctatgcc tctactacaaatctaagaat ttacgtaacg 1620 gttgcaggtg aacggatttt tgctggtcaa tttaacaaaacaatggatac cggtgaccca 1680 ttaacattcc aatcttttag ttacgcaact attaatacagcttttacatt cccaatgagc 1740 cagagtagtt tcacagtagg tgctgatact tttagttcagggaatgaagt ttatatagac 1800 agatttgaat tgattccagt tactgcaaca ctcgaggctgaatataatct ggaaagagcg 1860 cagaaggcgg tgaatgcgct gtttacgtct acaaaccaactagggctaaa aacaaatgta 1920 acggattatc atattgatca agtgtccaat ttagttacgtatttatcgga tgaattttgt 1980 ctggatgaaa agcgagaatt gtccgagaaa gtcaaacatgcgaagcgact cagtgatgaa 2040 cgcaatttac tccaagattc aaatttcaaa gacattaataggcaaccaga acgtgggtgg 2100 ggcggaagta cagggattac catccaagga ggggatgacgtatttaaaga aaattacgtc 2160 acactatcag gtacctttga tgagtgctat ccaacatatttgtatcaaaa aatcgatgaa 2220 tcaaaattaa aagcctttac ccgttatcaa ttaagagggtatatcgaaga tagtcaagac 2280 ttagaaatct atttaattcg ctacaatgca aaacatgaaacagtaaatgt gccaggtacg 2340 ggttccttat ggccgctttc agcccaaagt ccaatcggaaagtgtggaga gccgaatcga 2400 tgcgcgccac accttgaatg gaatcctgac ttagattgttcgtgtaggga tggagaaaag 2460 tgtgcccatc attcgcatca tttctcctta gacattgatgtaggatgtac agacttaaat 2520 gaggacctag gtgtatgggt gatctttaag attaagacgcaagatgggca cgcaagacta 2580 gggaatctag agtttctcga agagaaacca ttagtaggagaagcgctagc tcgtgtgaaa 2640 agagcggaga aaaaatggag agacaaacgt gaaaaattggaatgggaaac aaatatcgtt 2700 tataaagagg caaaagaatc tgtagatgct ttatttgtaaactctcaata tgatcaatta 2760 caagcggata cgaatattgc catgattcat gcggcagataaacgtgttca tagcattcga 2820 gaagcttatc tgcctgagct gtctgtgatt ccgggtgtcaatgcggctat ttttgaagaa 2880 ttagaagggc gtattttcac tgcattctcc ctatatgatgcgagaaatgt cattaaaaat 2940 ggtgatttta ataatggctt atcctgctgg aacgtgaaagggcatgtaga tgtagaagaa 3000 caaaacaacc aacgttcggt ccttgttgtt ccggaatgggaagcagaagt gtcacaagaa 3060 gttcgtgtct gtccgggtcg tggctatatc cttcgtgtcacagcgtacaa ggagggatat 3120 ggagaaggtt gcgtaaccat tcatgagatc gagaacaatacagacgaact gaagtttagc 3180 aactgcgtag aagaggaaat ctatccaaat aacacggtaacgtgtaatga ttatactgta 3240 aatcaagaag aatacggagg tgcgtacact tctcgtaatcgaggatataa cgaagctcct 3300 tccgtaccag ctgattatgc gtcagtctat gaagaaaaatcgtatacaga tggacgaaga 3360 gagaatcctt gtgaatttaa cagagggtat agggattacacgccactacc agttggttat 3420 gtgacaaaag aattagaata cttcccagaa accgataaggtatggattga gattggagaa 3480 acggaaggaa catttatcgt ggacagcgtg gaattactccttatggagga atag 3534 34 1177 PRT Artificial sequence HybridDelta-Endotoxin 34 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro TyrAsn Cys Leu 1 5 10 15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu ArgIle Glu Thr Gly 20 25 30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr GlnPhe Leu Leu Ser 35 40 45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly LeuVal Asp Ile Ile 50 55 60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala PheLeu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu PheAla Arg Asn Gln Ala 85 90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu TyrGln Ile Tyr Ala Glu 100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro ThrAsn Pro Ala Leu Arg Glu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp MetAsn Ser Ala Leu Thr Thr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln AsnTyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala AsnLeu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln ArgTrp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp LeuThr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp TyrAsn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp TrpVal Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260265 270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu275 280 285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser IleThr 290 295 300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser GlyHis Gln 305 310 315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro GluPhe Thr Phe Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro GlnGln Arg Ile Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr LeuSer Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn AsnGln Gln Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly ThrSer Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly ThrVal Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val ProPro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 Val Ser Met PheArg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala ProMet Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 Ile IleAla Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His 465 470 475 480Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490495 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500505 510 Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg515 520 525 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala GlyGlu 530 535 540 Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr GlyAsp Pro 545 550 555 560 Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile AsnThr Ala Phe Thr 565 570 575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val GlyAla Asp Thr Phe Ser 580 585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg PheGlu Leu Ile Pro Val Thr 595 600 605 Ala Thr Leu Glu Ala Glu Tyr Asn LeuGlu Arg Ala Gln Lys Ala Val 610 615 620 Asn Ala Leu Phe Thr Ser Thr AsnGln Leu Gly Leu Lys Thr Asn Val 625 630 635 640 Thr Asp Tyr His Ile AspGln Val Ser Asn Leu Val Thr Tyr Leu Ser 645 650 655 Asp Glu Phe Cys LeuAsp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 His Ala Lys ArgLeu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn 675 680 685 Phe Lys AspIle Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr 690 695 700 Gly IleThr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730735 Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740745 750 Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr755 760 765 Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser LeuTrp 770 775 780 Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu ProAsn Arg 785 790 795 800 Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu AspCys Ser Cys Arg 805 810 815 Asp Gly Glu Lys Cys Ala His His Ser His HisPhe Ser Leu Asp Ile 820 825 830 Asp Val Gly Cys Thr Asp Leu Asn Glu AspLeu Gly Val Trp Val Ile 835 840 845 Phe Lys Ile Lys Thr Gln Asp Gly HisAla Arg Leu Gly Asn Leu Glu 850 855 860 Phe Leu Glu Glu Lys Pro Leu ValGly Glu Ala Leu Ala Arg Val Lys 865 870 875 880 Arg Ala Glu Lys Lys TrpArg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895 Thr Asn Ile Val TyrLys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910 Val Asn Ser GlnTyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925 Ile His AlaAla Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940 Pro GluLeu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955 960Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970975 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980985 990 Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu995 1000 1005 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val ArgVal 1010 1015 1020 Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala TyrLys Glu 1025 1030 1035 Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu IleGlu Asn Asn 1040 1045 1050 Thr Asp Glu Leu Lys Phe Ser Asn Cys Val GluGlu Glu Ile Tyr 1055 1060 1065 Pro Asn Asn Thr Val Thr Cys Asn Asp TyrThr Val Asn Gln Glu 1070 1075 1080 Glu Tyr Gly Gly Ala Tyr Thr Ser ArgAsn Arg Gly Tyr Asn Glu 1085 1090 1095 Ala Pro Ser Val Pro Ala Asp TyrAla Ser Val Tyr Glu Glu Lys 1100 1105 1110 Ser Tyr Thr Asp Gly Arg ArgGlu Asn Pro Cys Glu Phe Asn Arg 1115 1120 1125 Gly Tyr Arg Asp Tyr ThrPro Leu Pro Val Gly Tyr Val Thr Lys 1130 1135 1140 Glu Leu Glu Tyr PhePro Glu Thr Asp Lys Val Trp Ile Glu Ile 1145 1150 1155 Gly Glu Thr GluGly Thr Phe Ile Val Asp Ser Val Glu Leu Leu 1160 1165 1170 Leu Met GluGlu 1175 35 20 DNA Artificial sequence Synthetic Oligonucleotide 35tgcaacactc gaggctgaat 20

What is claimed is:
 1. A modified Bacillus thuringiensis hybrid crystalprotein comprising an amino acid sequence at least 90% identical to SEQID NO:10, SEQ ID NO:12, SEQ ID NO:28, and SEQ ID NO:34.
 2. The proteinof claim 1, wherein: the hybrid protein exhibits increased insecticidalactivity against an insect, relative to the non-hybrid protein fromwhich it was engineered; and the insect is a member of an insect familyselected from the group consisting of Heliothis, Helicoverpa,Pectinophora, Spodoptera, and Earias.
 3. The protein of claim 2, whereinthe insect is a species selected from the group consisting of Heliothisvirescens, Helicoverpa zea, Helicoverpa armigera, Pectinophoragossypiella, Spodoptera exigua, Spodoptera frugiperda, Earias vitella,and Spodoptera litura.
 4. The protein of claim 1, wherein the protein isisolated from a Bacillus thuringiensis cell selected from the groupconsisting of EG11768, EG11063, EG11074, and EG11751.
 5. Apolynucleotide encoding the protein of claim
 1. 6. The polynucleotide ofclaim 5, wherein the polynucleotide hybridizes under high stringencyconditions with a sequence which is or is complementary to the sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:27, and SEQ ID NO:33.
 7. The polynucleotide of claim 5, wherein: thepolynucleotide encodes a hybrid δ-endotoxin protein having increasedinsecticidal activity against an insect, relative to the non-hybridprotein from which it was engineered; and the insect is a member of aninsect family selected from the group consisting of Heliothis,Helicoverpa, Pectinophora, Spodoptera, and Earias.
 8. The polynucleotideof claim 7, wherein the insect is a species selected from the groupconsisting of Heliothis virescens, Helicoverpa zea, Helicoverpaarmigera, Pectinophora gossypiella, Spodoptera exigua, Spodopterafrugiperda, Earias vitella, and Spodoptera litura.
 9. The polynucleotideof claim 5, wherein the polynucleotide comprises a sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:27,and SEQ ID NO:33.
 10. The polynucleotide of claim 5, further comprisinga recombinant vector.
 11. The polynucleotide of claim 5, wherein thepolynucleotide is operatively linked to a promoter.
 12. A recombinanthost cell comprising the polynucleotide of claim
 11. 13. The recombinanthost cell of claim 12, wherein the polynucleotide comprises a sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:27, and SEQ ID NO:33.
 14. The recombinant host cell of claim 12,wherein the host cell is an E. coli, B. thuringiensis, B. subtilis, B.megaterium, or a Pseudomonas spp. cell.
 15. The recombinant host cell ofclaim 14, wherein said B. thuringiensis cell selected from the groupconsisting of EG11768, EG11063, EG11074, and EG11751.
 16. Therecombinant host cell of claim 12, defined further as being a eukaryoticcell.
 17. The recombinant host cell of claim 12, further defined as aplant cell.
 18. The recombinant host cell of claim 17, wherein the plantcell is a corn, wheat, oat, barley, cotton, soybean, maize, rye, turfgrass, pasture grass, vegetable, berry, fruit, tree, or ornamental plantcell.
 19. A method of using a DNA segment that encodes a δ-endotoxincrystal protein or peptide, comprising the steps of: (a) introducing thepolynucleotide of claim 11 into a host cell; (b) culturing the host cellunder conditions effective to allow expression of the encoded crystalprotein or peptide; and (c) collecting the crystal protein or peptidethus produced.
 20. A composition comprising the crystal protein ofclaim
 1. 21. The composition of claim 21, wherein the crystal protein isencoded by a sequence comprising SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:27, or SEQ ID NO:33.
 22. A method of controlling insect pestscomprising contacting an insect with a Bacillus thuringiensis crystalprotein encoded by the polynucleotide of claim
 5. 23. The method ofclaim 22, wherein the protein is produced by a transgenic plant, and thetransgenic plant has incorporated into its genome a polynucleotide thathybridizes under high stringency conditions with a sequence which is oris complementary to the sequence selected from the group consisting ofSEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:27, and SEQ ID NO:33.
 24. Themethod of claim 22, wherein the protein is produced by a recombinanthost cell, and the recombinant host cell has incorporated into itsgenome a polynucleotide that hybridizes under high stringency conditionswith a sequence which is or is complementary to the sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:27,and SEQ ID NO:33.
 25. The method of claim 22, wherein the protein isencoded by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:27, or SEQ ID NO:33. 26.The method of claim 22, wherein the protein comprises SEQ ID NO:10, SEQID NO:12, SEQ ID NO:28, and SEQ ID NO:34.
 27. A transgenic plant havingincorporated into its genome the polynucleotide of claim
 11. 28. Thetransgenic plant of claim 27, wherein said polynucleotide comprises SEQID NO:9, SEQ ID NO:11, SEQ ID NO:27, or SEQ ID NO:33.
 29. A progeny ofthe plant of claim
 28. 30. A seed from the plant of claim
 28. 31. A seedfrom the progeny of claim
 30. 32. A plant from the seed of claim 31.